Silver nanoparticle-induced developmental inhibition of Drosophila melanogaster accompanies disruption of genetic material of larval neural stem cells and non-neuronal cells.

A few studies had determined the effects of silver nanoparticles on the development of Drosophila melanogaster. However, none had addressed its genotoxic effects on specific larval cells of the fly in details. This study was conducted to determine the effects of silver nanoparticle on the development of D. melanogaster with simultaneous evaluation of its genotoxic potential on specific larval cell types that play important roles in immunological defenses as well as growth and development. Five male and five female flies were maintained in standard Drosophila melanogaster culture medium containing varying concentrations of silver nanoparticles, i.e., 25, 50, 100, 200, and 300 mg/l with control culture medium containing no nanoparticle. Total time needed for stage-specific development, population yield, and genotoxic effects on third instar larval polytene chromosomes, hemocytes, and neuroblasts was determined. Body pigmentation of pupae and young adults was examined visually. In comparison with control, silver nanoparticles dose dependently inhibited the metamororphosis and population yields of pupae and young adults of Drosophila melanogaster. Every concentration of the nanoparticles inhibited pupa to adult conversion, with huge reduction under the influence of nanoparticle concentration of 100 mg/ml and above. Developmental inhibition was accompanied by dose-dependent and significant structural aberrations of larval polytene chromosomes and deformities of hemocytes and neuroblasts. Pupae and young adults also exhibited gradual discoloration of body with the increase in exposure to nanoparticle concentration.

Hydroxyurea responses in clinically varied beta, HbE-beta thalassaemia and sickle cell anaemia patients of Eastern India.

The haematological and clinical response to hydroxyurea was estimated in HbE-beta, beta thalassaemia and sickle cell anaemia patients of Eastern India, with variable clinical severity and transfusion requirement to determine whether hydroxyurea can help these patients to maintain their steady haemoglobin level without blood transfusions. Three hundred patients (189 HbE-beta thalassaemia, 95 beta thalassaemia and 16 other haemoglobinopathies including sickle cell anaemia) were selected for hydroxyurea therapy and were followed up for 48-60 months. Results suggest significant response to hydroxyurea therapy in 19 beta and 99 HbE-beta patients in the transfusion-dependent group (GR-I). All of them became transfusion-independent while on hydroxyurea therapy. The majority of responding patients were IVS1-5(G-C) in one of their alleles in HbE-beta cases (83 out of 119). Though IVS1-5(G-C) was found to be the commonest mutation in our selected patients, the mutational background of the patients does not found to have any significant correlation with the response category towards hydroxyurea as per the results observed in our study. But, the drug works pretty well in most of the transfusion-dependent patients, as these patients were withdrawn from regular blood transfusion. At the same time, partial or no response to the drug hydroxyurea was also recorded in our study.

Population Screening and Prevention Strategies for Thalassemias and other Hemoglobinopathies of Eastern India: Experience of 18,166 cases.

We evaluated population screening programs (1999-2011), conducted by the Thalassaemia Foundation, Kolkata, India, for the first time in Eastern India in different districts of West Bengal, for prevention of thalassemia comprising screening of heterozygotes and ß-thalassemia intermedia (ß-TI) cases [ß(+), ß(++), ß(0)/ß(+), ß(E)/ß(E) (codon 26 or HBB: c.79G > A), Hb-E-ß-thalassemia (Hb E-ß-thal)]. Among 18,166 cases, we found 2092 heterozygotes and 2245 ß-TI individuals (who had no information about their disorders). Results were evaluated with standard hematological analyses including erythrocyte indices, hemoglobin (Hb) typing and quantification. Participants were divided into five groups (children, pre-marriage cases, pre-pregnancy cases, affected family members, pregnant women). The objectives of this evaluation were to fix cut-off values of red blood cells (RBCs), mean corpuscular volume (MCV), mean corpuscular Hb (MCH), red blood cell distribution width (RDW) and Hb A2, as the standard World Health Organization (WHO) guidelines were not strictly followed in mass-scale screening programs. We have observed many dilemmas in considering the status of the thalassemia subject, due to presence of some other clinical conditions such as iron deficiency anemia, α-thalassemia (α-thal), δ-thalassemia (δ-thal), clinically silent Hb variants, and some cases of non hemoglobinopathies (such as pregnancy) along with thalassemia. The MCV values varied greatly in different conditions of hemoglobinopathies, whereas MCH provided a more stable measurement. We found an MCH value of <27.0 pg is a suitable cut-off point for screening in this population. Participants with an MCH of <27.0 pg should be investigated further to confirm or exclude a diagnosis of ß-thal trait.

Mutation Spectrum of ß-Thalassemia and Other Hemoglobinopathies in Chittagong, Southeast Bangladesh.

Thalassemia is one of the most common autosomal recessive blood disorders in the world. It shows a variety of clinical expression, starting from asymptomatic to severe blood transfusion dependence. More than 500 alleles have been characterized in or around the ß-globin region. Moreover, most geographical regions have their own characteristics, frequency and availability of these alleles, predominantly circulating within the communities present in that particular region. In this study, we explored the spectrum of ß-thalassemia (ß-thal) alleles present in Chittagong, Southeast Bangladesh. This study comprises ß-thal and Hb E (HBB: c.79 G > A) patients from in and around the area of Chittagong. Not only exploring the complete ß-globin mutation spectrum of the area, but we also tried to look at the origin of the mutated alleles. The ß-thal mutations of Bangladesh show a relatively wide spectrum of alleles, which further demonstrates the heterogeneity of the disease in this country. Although our study showed that the majority of the mutations have their origin in neighboring countries such as India, countries of Southeast Asia, Pakistan, etc., some unusual alleles do not originate in neighboring countries and put a little more diversity in the overall spectrum of ß-thal-specific alleles. Overall, this study demonstrates the mutation spectrum related to ß-thal in Chittagong, Southeast Bangladesh.

Mutational spectrum of thalassemias and other hemoglobinopathies in West Bengal, Eastern India.

Thalassemia, an autosomal recessive blood disease, shows a variety of clinical expression in terms of asymptomatic to severe blood transfusion dependence. More than 500 alleles have been characterized in or around the ß-globin region. Most of the geographical regions have their own characteristic alleles that predominantly circulate within the communities present in that particular region. In this article, we try to throw some light to explore the spectrum of ß-thalassemia (ß-thal) alleles present in West Bengal, the eastern part of India. This study comprises thalassemia carriers and diseased persons from different districts of West Bengal. We not only explored the complete mutational spectrum of this state but we also tried to fix the critical range of the values of different hematological parameters [Hb A2, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH)] for the heterozygotes or carriers of ß-thal with the same mutational background. At the same time, we also tried to evaluate the maximum weighted frequency of these parameters for the heterozygotes or carriers of ß-thal with the same mutational background, so that by observing these cut-off values of standard hematological parameters, we were able to predict the carrier or diseased status for mass scale screening and also try to correlate the values of these parameters with different combinations of ß-thal mutation-specific alleles that can be more informative in mass scale (carrier) screening.

Benzaldehyde-induced developmental genotoxicity triggers both neural and non-neuronal cells including the cells of immunity in Drosophila melanogaster.

Although benzaldehyde, an aromatic aldehyde, has been declared safe for uses in food, conflicting reports exist regarding its genotoxic and cytotoxic potentials in organisms. Our present study is the first attempt to evaluate the effects of exposure of benzaldehyde on the entire course of development of a eukaryote model organism, Drosophila melanogaster. Total time required for the initial appearance of the third instar larvae, pupae and adults increased dose dependently with the increasing dietary concentration of benzaldehyde. Exposure of flies to each concentration of benzaldehyde caused dose-dependent and significant reductions in the population of pupae and young adults of the fly. Developmental inhibition was associated with dose dependent and significant structural aberrations of larval polytene chromosomes like ectopic pairing, inversion, fusion, etc., and deformities of hemocytes and neuroblasts and death of hemocytes. As much as 34% (SD ± 1.76)-52% (SD ± 1.7) and 18% (SD ± 2.5)-40% (SD ± 3.38) hemocytes and neuroblasts, respectively, underwent nuclear deformations in response to dietary exposures of flies to BA 100-1000 mg/l. Moreover, 16% (SD ± 0.52)-31% (SD ± 1.97) and 19% (SD ± 0.3)-33% (SD ± 1.78) hemocyte mortalities in response to BA 100-1000 mg/l were determined by two cell viability assays. Thus our study revealed that benzaldehyde was genotoxic to Drosophila melanogaster larvae that might be responsible for larval cell death and their subsequent developmental retardation. As this fly possesses substantial genetic homology with human, possibility of developmental inhibition of the later due to exposure of this chemical during pregnancy may not be ruled out.

F-cell levels are altered with erythrocyte density in sickle cell disease.

Lighter cells from density fractionated erythrocytes of sickle cell disease (SCD) patients carry higher amount of externalized phosphatidylserine (PS) and cell surface glycophorins compared to the denser counterparts. Further analysis also revealed that the denser cells contained higher levels of fetal hemoglobin (HbF) compared to the lighter cells, supported by the presence of larger number of F-cells in these populations. In this report, we have found direct evidence on the higher survival of the HbF rich erythrocytes in SCD.

Impacts of dietary exposure to sodium or potassium salts of nitrate and nitrite on the development of Drosophila melanogaster.

The effects of four food additives, namely sodium nitrite (NaNO2), sodium nitrate (NaNO3), potassium nitrite (KNO2), and potassium nitrate (KNO3), on animal development were evaluated by using Drosophila melanogster, a model organism. Adult male and female flies were allowed to breed in culture medium, each containing one of 4 concentrations, i.e.10, 20, 30 or 40 mM of the above mentioned salts. The concentration of 40 mM, NaNO2 and KNO2 completely arrested the development of the flies. Of the different concentrations of the four salts tested, exposure of flies to 30 mM NaNO2 exhibited only significant delays in the initial appearances of third instar larvae, pupae and young adults, along with huge reduction in the number of pupae and young adults compared to controls. Rearrangements like inversions, deletion looping, regional shrinking, as well as highly enlarged puffing, etc. were also observed in the polytene chromosomes of the third instar larvae exposed to 30 mM NaNO2. Developmental outcomes of the flies exposed to varying concentrations of NaNO3 and KNO3 did not differ significantly from the controls. Owing to the extensive genetic homology between Drosophila and human and the successful uses of this fly as models in developmental and toxicological studies, we speculate that the experimental results exhibited by this organism in our study strongly advocate for abstaining from the dietary use of NaNO2 and KNO2 during human pregnancies to avoid possible negative developmental outcomes.

Inositol hexa phosphoric acid (phytic acid), a nutraceuticals, attenuates iron-induced oxidative stress and alleviates liver injury in iron overloaded mice.

Inositol hexa phosphoric acid (IP6) or Phytic acid, a natural antioxidant of some leguminous plants, known to act as a protective agent for seed storage in plants by suppressing iron catalyzed oxidative process. Following the same mechanism, we have tested the effect of IP6 on iron overloaded in vitro oxidative stress, and studied it's in vivo hepatoprotective ability in iron-dextran (injection)-induced iron overloaded liver injury in mice (intraperitoneal). Our results showed that IP6 had in vitro iron chelation (IC50 38.4µg/ml) activity, with the inhibition of iron-induced lipid peroxidation (IC50 552µg/ml), and deoxyribose sugar degrading hydroxyl radicals (IC50 448.6µg/ml). Oral administration of IP6 (0-200mg/kg) revealed significant decrease in biochemical markers such as serum iron, total iron binding, serum ferritin and serum enzymes. Histopathology of liver stained with hematoxylin-eosin and Prussian blue showed reduced hepatocellular necrosis, ballooning and inflammation, indicating the restoration of normal cellular integrity. Interestingly, the IP6 was found to down-regulate the mRNA expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1ß, and IL-6 in iron overloaded liver tissues. Thus, we provide an insight that IP6, a natural food component, can serve as an iron chelator against iron overload diseases like Thalassemia, and also as a dietary hepatoprotective supplement.

A FACS Based Case Study on Two HbE-ß Thalassaemia Members of a Family, Having Similar Mutational Background.

In this report we have tried to explain the reasons behind the difference in the pattern of transfusion requirement between two members of a family with similar ß-globin mutation. The father and younger son both are HbE-ß, but the father never had transfusion, whereas the younger son takes transfusion monthly. Mother and the elder son are HbEE without any history of transfusion. ß-globin mutations of all family members were determined by ARMS-PCR. These were reconfirmed by direct sequencing of ß-globin gene. Father and younger son were found to be Cod 26 (G-A)/IVS 1-5 (G-C), whereas mother and elder son were found to be Cod 26 (G-A)/Cod 26 (G-A). XmnI sequencing also revealed that all members of the family were CC. Then, flow cytometry study of red blood cells (RBCs) was performed to measure the oxidative stress of the RBCs. This study was also done on the light and dense fractions of the RBC population of the father and younger son. It was seen that the younger son suffers severe oxidative stress, which can be explained by his higher transfusion requirement. From our work, we have established the importance of taking oxidative stress of RBCs into consideration to explain the clinical manifestation and progression of haemoglobin related diseases like thalassaemia.

Novel balanced chromosomal translocations in females with recurrent spontaneous abortions: Two case studies.

Two couples with a history of recurrent pregnancy losses were referred to the Institute of Genetic Medicine and Genomic Science for cytogenetic evaluation. Chromosomal analysis of the phenotypically normal couples was done to investigate whether there are any new chromosomal abnormalities present in either of the couples caused recurrent pregnancy losses. Clinical and hormonal profile of the couples revealed normal parameters. The ultrasound scan of the females showed normal uterine and ovarian structures. Chromosomal analysis of the couples revealed normal 46, XY karyotypes in the both the male partners, and novel balanced reciprocal translocations 46, XX, t (5;8) (q35.3;q24.23) and 46, XX, t (4;13) (q12;q14) chromosomal constitutions in the female partners. Further, corroboration of the chromosome abnormalities was carried out by high resolution banding analysis. Unique and novel balanced reciprocal translocations were reported as an original investigation in two female partners from two different unrelated families both with the history of recurrent pregnancy losses.

Clinical presentation of two ß-thalassemic Indian patients with 1p36 deletion syndrome: Case report.

Here, we present two thalassemic patients (one male and one female), having unusual clinical phenotypes. Both had mental retardation in which one was associated with microcephaly and other had congenital cataract. They were referred to our institute for clinical evaluation and cytogenetic testing. Both patients were tested for presence of abnormal hemoglobin by high performance liquid chromatography and found to be thalassemic. Their ß-globin mutation was also determined by amplification refractory mutation system-polymerase chain reaction. The male patient was found to have intervening sequence 1-5 (G-C)/+, indicating ß-thalassemia trait and the female was found to have Cod 26 (G-A)/IVS 1-5 (G-C), indicating hemoglobin E-ß thalassemia. Their cytogenetic analysis of blood lymphocytes were studied with high-resolution GTG-banding analysis by using chromosome profiling (Cyto-vision software 3.6) on their chromosomes. Results revealed 46,XY,del(1)(p36.21) in the male and 46,XX,del(1)(p36.3) in the female. Their genotype variation showed (based on genome browser) significant gene loss which probably leads to marked phenotype variation. We believe, thalassemia with mental retardation associated with microcephaly and congenital cataract, both having loss in chromosome 1, p36 position, is reported probably first time from India. This report will definitely enlighten all concerns and add to the information in growing literature.

Differential regulation of redox proteins and chaperones in HbEß-thalassemia erythrocyte proteome.

PURPOSE: In (hemoglobin, Hb) HbEß-thalassemia, HbE (ß-26 Glu→Lys) interacts with ß-thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEß-thalassemic patients compared to normal. EXPERIMENTAL DESIGN: We have used 2-DE and MALDI-MS/MS-based techniques to investigate the differential proteome profiling of membrane and Hb-depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEß-thalassemia patients and normal volunteers. RESULTS: Our study showed that redox regulators such as peroxiredoxin 2, Cu-Zn superoxide dismutase and thioredoxin and chaperones such as α-hemoglobin stabilizing protein and HSP-70 were upregulated in HbEß-thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin-based junctional complex in the membrane skeleton of HbEß-thalassemic erythrocytes upon detection of low molecular weight fragments of ß-spectrin and decrease in ß-actin and dematin content. CONCLUSION AND CLINICAL RELEVANCE: We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.

Enhanced oxidative cross-linking of hemoglobin E with spectrin and loss of erythrocyte membrane asymmetry in hemoglobin Ebeta-thalassemia.

Oxidative stress to the erythrocytes is associated with formation of large molecular complexes of hemoglobin and the skeletal protein, spectrin. In this work, such complexes are formed with hemoglobin mixtures isolated from patients suffering from HbEbeta-thalassemia with elevated levels of the HbE and purified erythroid spectrin in the presence of hydrogen peroxide. The complexes are separated on 4% SDS-PAGE and analyzed by densitometry. The results indicate enhanced formation of complexes with higher amounts of HbE, the most common hemoglobin variant prevalent in Southeast Asia. The binding affinity of spectrin with hemoglobin, in the absence of hydrogen peroxide, was found to increase with hemoglobin mixtures enriched with HbE. The presence of ATP was also found to decrease the overall yield of such complexes. Flow cytometric measurements of phosphatidylserine on the red cell surface also showed a lower degree of membrane asymmetry in HbEbeta-thalassemic patients than in normal subjects. The present work shows enhanced formation of high molecular weight cross-linked complexes of hemoglobin derivatives with erythroid spectrin in HbEbeta-thalassemia.