Hypofractionated radiotherapy in young versus older women with breast cancer: a retrospective study from India

BackgroundYoung women with breast cancer (BC) are not represented in the trials on hypofractionation. In this study we compared outcomes in young patients with BC to their older counterparts treated with hypofractionated radiotherapy (RT) in a regional cancer centre in India.Materials and methodsBetween January 1990 to December 2010, women with BC, treated with hypofractionated RT dose of 35–40 Gy/15#/3 weeks were divided into two groups, ≤ 35 years and > 35 years. Outcomes compared were locoregional recurrence rate (LRR), locoregional recurrence-free survival (LRRFS), disease-free survival (DFS), overall survival (OS) and toxicities. LRRFS, DFS and OS were estimated using the Kaplan-Meier method.ResultsOf total 2244 patients, 359 were ≤ 35 years of age and 1885 were > 35 years. Patient and disease characteristics were comparable between the two groups, except that comorbidities were significantly higher in the > 35 years age group, more patients aged ≤ 35 years had nodal N3 disease, received chemotherapy and RT to internal mammary nodes and more patients in the > 35 years group received hormonal therapy. Median follow up was 10 years (range 1–30 years). LRR and distant metastases were comparable between the two groups. However, synchronous LRR and distant metastases were significantly higher in the ≤ 35 years group 18 (5.1%) as compared to the > 35 years group 39 (2.1%) with p = 0.018. Estimated 10-year LRRFS, DFS and OS were 92% vs. 94% (p = 0.95), 68% vs. 73%(p = 0.058) and 78% vs. 76% (p = 0.10) in ≤ 35 years and > 35 years, respectively. OS for stage 1 was comparable between the two groups. However, for stage 2 and 3 it was 77% vs. 82% (p = 0.048) and 53% vs. 62% (p = 0.045) in the ≤ 35 years and > 35 years group, respectively. Acute and late toxicity were similar in the two groups.ConclusionYoung BC patients had higher LRR and distant metastases. LRRFS, DFS and toxicities were comparable between the two groups. However, OS was poorer in young BC patients with stage 2 and 3 disease.     

Kalirin-7 controls activity-dependent structural and functional plasticity of dendritic spines

Activity-dependent rapid structural and functional modifications of central excitatory synapses contribute to synapse maturation, experience-dependent plasticity, and learning and memory and are associated with neurodevelopmental and psychiatric disorders. However, the signal transduction mechanisms that link glutamate receptor activation to intracellular effectors that accomplish structural and functional plasticity are not well understood. Here we report that NMDA receptor activation in pyramidal neurons causes CaMKII-dependent phosphorylation of the guanine-nucleotide exchange factor (GEF) kalirin-7 at residue threonine 95, regulating its GEF activity, leading to activation of small GTPase Rac1 and rapid enlargement of existing spines. Kalirin-7 also interacts with AMPA receptors and controls their synaptic expression. By demonstrating that kalirin expression and spine localization are required for activity-dependent spine enlargement and enhancement of AMPAR-mediated synaptic transmission, our study identifies a signaling pathway that controls structural and functional spine plasticity.     

Single cell gel electrophoresis assay (comet assay): its importance in human biology

The estimation of genetic instability by direct extent of DNA damage and repair is an important aspect of studies on mutagenesis, carcinogenesis, aging and evolution. Different methods have been introduced from time to time in an effort to meet this need. Single cell gel electrophoresis (SCGE) assay is a new, simple and sensitive method of evaluating DNA damage and repair at individual cell level. This assay can be performed on extremely small number of cells and results can be obtained within a relatively short time. The SCGE assay has the potential to play an important role not only in the understanding of some of the fundamental aspects of human biology but also can be helpful in many practical ways. For reprint requests.     

Changes in cytosolic Ca2+ levels correspond to fluctuations of lactate levels in crosstalk of astrocyte-neuron cell lines

Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with CSD1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.     

Antiangiogenic and proapoptotic activity of a novel glycoprotein from U. indica is mediated by NF-kappaB and Caspase activated DNase in ascites tumor model

We have identified a novel glycoprotein from Urginea indica bulbs with potent in vivo antitumor activity against growth of an ascites tumor, mouse mammary carcinoma. In this paper we report characterization of a 29 kDa glycoprotein from U. indica and demonstrate the mechanism of antiangiogenic and proapoptotic activity. N-terminal sequence of the high performance liquid chromatography (HPLC) pure glycoprotein showed sequence homology to an extent of 40-50% with known angiogenesis inhibitor and apoptosis-inducing protein from C. elegans and G. gallus respectively. Our results on antiangiogenic property of the glycoprotein include inhibition of in vivo angiogenesis assays, decreased micro vessel density count and CD31 antigen staining in 29 kDa glycoprotein treated mice peritoneum. In vitro inhibition of vascular endothelial growth factor induced proliferation of human umbilical vein endothelial cells (HUVECs) by the glycoprotein further supports its antiangiogenic activity. The mechanism of antiangiogenesis involved inhibition of translocation of nuclear factor kappa B to the nucleus resulting in decreased expression of vascular endothelial growth factor gene as is demonstrated by our results on quantification of vascular endothelial growth factor levels in the glycoprotein treated tumor bearing mice. Our results on activation of Caspase-3 with concomitant translocation of caspase activated DNase to the tumor cell nuclei resulting in DNA fragmentation induced by the glycoprotein in vivo clearly demonstrated a parallel proapoptotic activity of the glycoprotein.     

The environmental toxin arsenite induces tau hyperphosphorylation

Abnormally hyperphosphorylated tau polymers known as paired helical filaments constitute one of the major characteristic lesions that lead to the demise of neurons in Alzheimer's disease. Here, we demonstrate that the environmental toxin arsenite causes a significant increase in the phosphorylation of several amino acid residues (Thr-181, Ser-202, Thr-205, Thr-231, Ser-262, Ser-356, Ser-396, and Ser-404) in tau, which are also hyperphosphorylated under pathological conditions. Complementary phosphopeptide mapping revealed a dramatic increase in the (32)P-labeling of many peptides in tau following arsenite treatment. Although arsenite activates extracellular-signal regulated kinases-1/-2 and stress-activated protein kinases, these enzymes did not contribute to the arsenite-increased phosphorylation, nor did they appear to normally modify tau in vivo. Tau phosphorylation induced by arsenite did not involve glycogen synthase kinase-3 or protein phosphatase-1 or -2, but the activity responsible for tau hyperphosphorylation could be inhibited with the protein kinase inhibitor roscovitine. The effects of arsenite on the phosphorylation of some tau mutations (DeltaKappa280, V337M, and R406W) associated with frontal-temporal dementia with parkinsonism linked to chromosome 17 was analyzed. The unchallenged and arsenite-induced phosphorylation of some mutant proteins, especially R406W, was altered at several phosphorylation sites, indicating that these mutations can significantly affect the structure of tau in vivo. Although the major kinase(s) involved in aberrant tau phosphorylation remains elusive, these results indicate that environmental factors, such as arsenite, may be involved in the cascade leading to deregulation of tau function associated with neurodegeneration.     

The zinc dyshomeostasis hypothesis of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia in the elderly. Hallmark AD neuropathology includes extracellular amyloid plaques composed largely of the amyloid-β protein (Aβ), intracellular neurofibrillary tangles (NFTs) composed of hyper-phosphorylated microtubule-associated protein tau (MAP-tau), and microtubule destabilization. Early-onset autosomal dominant AD genes are associated with excessive Aβ accumulation, however cognitive impairment best correlates with NFTs and disrupted microtubules. The mechanisms linking Aβ and NFT pathologies in AD are unknown. Here, we propose that sequestration of zinc by Aβ-amyloid deposits (Aβ oligomers and plaques) not only drives Aβ aggregation, but also disrupts zinc homeostasis in zinc-enriched brain regions important for memory and vulnerable to AD pathology, resulting in intra-neuronal zinc levels, which are either too low, or excessively high. To evaluate this hypothesis, we 1) used molecular modeling of zinc binding to the microtubule component protein tubulin, identifying specific, high-affinity zinc binding sites that influence side-to-side tubulin interaction, the sensitive link in microtubule polymerization and stability. We also 2) performed kinetic modeling showing zinc distribution in extra-neuronal Aβ deposits can reduce intra-neuronal zinc binding to microtubules, destabilizing microtubules. Finally, we 3) used metallomic imaging mass spectrometry (MIMS) to show anatomically-localized and age-dependent zinc dyshomeostasis in specific brain regions of Tg2576 transgenic, mice, a model for AD. We found excess zinc in brain regions associated with memory processing and NFT pathology. Overall, we present a theoretical framework and support for a new theory of AD linking extra-neuronal Aβ amyloid to intra-neuronal NFTs and cognitive dysfunction. The connection, we propose, is based on β-amyloid-induced alterations in zinc ion concentration inside neurons affecting stability of polymerized microtubules, their binding to MAP-tau, and molecular dynamics involved in cognition. Further, our theory supports novel AD therapeutic strategies targeting intra-neuronal zinc homeostasis and microtubule dynamics to prevent neurodegeneration and cognitive decline.     

Factors affecting genetic transformation and shoot organogenesis of Bacopa monnieri (L.) Wettst

Efficient Agrobacterium tumefaciens mediated T-DNA delivery and subsequent shoot organogenesis has been achieved from Bacopa monnieri. Various factors influenced T-DNA delivery as evident from transient GUS assay. The transient GUS expression was significantly higher (97.7 %) in explants that were pre-cultured before bacterial infection on medium supplemented with 100 μM acetosyringone. Incorporation of acetosyringone into the co-cultivation medium also enhanced transient GUS activity. Explant injury with carborundum paper, co-cultivation period of 2 days and a bacterial density of 0.4 OD₆₀₀ showed higher transient GUS expression. Following co-cultivation, shoot organogenesis was achieved from leaf segments on basal Murashige and Skoog medium containing 58 mM sucrose. Supplementation of antibiotics (cefotaxime or carbenicillin) at > 250 μg/ml into the medium significantly promoted shoot organogenesis from leaf explants (71.5 % in control and > 83.0 % on medium containing 500 μg/ml of carbenicillin or cefotaxime). Stable transformation of regenerated shoots was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to reporter gene (uidA) and selection marker gene (nptII). The expression level of nptII gene in independent transgenic lines was studied using quantitative real time-PCR. Stable transformed shoots after rooting were successfully established in the pots.     

Data on the effect of sub-epithelial connective tissue graft with hydroxyl-apatite for furcation treatment in Indians

Grade II furcation defect has a great potential for regeneration. Attempts have been made to regenerate lost periodontium in furcation defects through variety of approaches. A total of 22 patients with Grade II furcation defect were examined. The patients were sequentially grouped into three groups namely, (Group A) subepithelial connective tissue graft as a barrier membrane, (Group B) bovine bone graft material Bio-OssTM and (Group C) with the combination of the two. The treatment modalities show that there was a strong improvement in the resolution of the grade II furcation defects. The reduction in horizontal furcation defect measurement was 20.63% for group A. The change was found to be moderate (p=0.023*). The % change is 20.97% for group B and it is statistically significance (p = 0.001**). The % change was 20.78% in Group C with a good statistical significance (p=0.003**). Complete obliteration of the defect clinically and radio-graphically was observed for grade II furcation defect. Thus, the effect of sub-epithelial connective tissue graft with hydroxyl-apatite for furcation treatment in Indians is documented in this report.