Lymphangiogenesis is required for pancreatic islet inflammation and diabetes

Lymphangiogenesis is a common phenomenon observed during inflammation and engraftment of transplants, but its precise role in the immune response and underlying mechanisms of regulation remain poorly defined. Here we showed that in response to injury and autoimmunity, lymphangiogenesis occurred around islets and played a key role in the islet inflammation in mice. Vascular endothelial growth factors receptor 3 (VEGFR3) is specifically involved in lymphangiogenesis, and blockade of VEGFR3 potently inhibited lymphangiogenesis in both islets and the draining LN during multiple low-dose streptozotocin (MLDS) induced autoimmune insulitis, which resulted in less T cell infiltration, preservation of islets and prevention of the onset of diabetes. In addition to their well-known conduit function, lymphatic endothelial cells (LEC) also produced chemokines in response to inflammation. These LEC attracted two distinct CX3CR1(hi) and LYVE-1(+) macrophage subsets to the inflamed islets and CX3CR1(hi) cells were influenced by LEC to differentiate into LYVE-1(+) cells closely associated with lymphatic vessels. These observations indicate a linkage among lymphangiogenesis and myeloid cell inflammation during insulitis. Thus, inhibition of lymphangiogenesis holds potential for treating insulitis and autoimmune diabetes.     

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 g/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 g/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.     

Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction

Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7-10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. d-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes.     

Improving the yield and quality of DNA isolated from white-rot fungi

A new simple method used to eliminate polysaccharides that cause problems during DNA isolation was established for 6 different white-rot fungi using 1% hexadecyltrimethylammonium bromide (CTAB) as wash buffer and followed by centrifugation. Variation in the DNA yield and quality was ascertained using precipitating agents, detergents and cell-wall-hydrolyzing chitinase. Considerable amount of exopolysaccharides from fungal biomass was removed with the use of 1% CTAB wash buffer followed by centrifugation. The DNA varied in terms of yield and quality. For the DNA extraction use of 2% SDS in extraction buffer worked best for Pycnoporus cinnabarinus, Cyathus bulleri, Cyathus striatus and Cyathus stercoreus, while 2% CTAB worked best for Phanerochaete chrysosporium and Pleurotus ostreatus. Elimination of phenol and use of absolute ethanol for precipitating DNA resulted in good yield and quality of DNA. This DNA was amenable to restriction endonuclease digestion.     

Mechanisms of C Sequestration in Soils of Latin America

Carbon (C) sequestration, defined as the process whereby atmospheric CO₂ is transferred into a long-lived C pool, is an important issue not only in the scientific community but also in the society at large because of its potential role in off-setting fossil fuel emissions. Through photosynthesis this C is stored in plants and through decomposition, trunks, branches, leaves and roots are incorporated in the soil via the action of different soil organisms, i.e., bacteria, fungi and invertebrates. This, together with the C exudates from roots that are utilized by microbial populations, constitutes the natural pathways of incorporating biomass-C into the soil. The amount of C stored in terrestrial ecosystems is the third largest among the global C pools. Soil organic carbon (SOC) up to 3 m is 2,344 Pg C (1 Petagram = 10¹⁵ g), and the SOC pool in tropical soils is approximately 30% of the global pool. Abiotic factors, which moderate C sequestration in soils are clay content, mineralogy, structural stability, landscape position, and soil moisture and temperature regimes. On the other hand, biotic factors involved in soil C sequestration are determined by the activities of soil organisms. However, models do not include the formation, stabilization and lifespan of the aggregates that have been biologically produced, including roots. This is not only due to the lack of studies on this subject, but also to overlooking the role of soil organisms in soil aggregation. Furthermore, there is a lack of comprehensive knowledge regarding the processes that control dissolved organic carbon (DOC) fluxes in soils and its role in the global budget of C sequestration. The boundaries of ecosystems are not considered in the studies of the subject, as it may be the case for terrestrial C sequestration, since the borders around the sites under study constitute pathways for the flow of C between sites and through the landscape. The concentrations of DOC in deep soil horizons and the contribution to DOC fluxes (exports) are relatively small, from 4 to 37 g DOC m^?2 yr^?1 retained in the mineral subsoil. In South America, although substantial research has been done under different ecosystems and land use systems in some countries, like Brazil, Colombia, Argentina, there is a need to conduct more studies with agreed standard methodologies in natural ecosystems and agricultural systems, and in other areas of Central America few studies have been undertaken to date. The principal objective of this review was to address the main mechanisms that determine SOC and SIC sequestration in soils of Latin America, and include: physical aggregate protection, SOC-clay interaction, DOC transport, bioturbation by soil organisms, and the formation of secondary carbonates. All of these mechanisms are generally explained by physical and chemical processes. In contrast, this review takes a soil ecological approach to describe the mechanisms listed above.     

Activated mouse T cells downregulate, process and present their surface TCR to cognate anti-idiotypic CD4+ T cells

The ability of activated T cells to present foreign antigens through the MHC class II pathway has been shown in the case of human, rat and mouse T cells. In the present study, the ability of activated T cells to present their endogenous TCR in association with MHC class II molecules to CD4+ T cells was shown. Upon activation mouse T cells downregulate their surface TCR, which are degraded into peptides in endosomal/lysosomal compartments. The idiopeptides (peptides derived from the variable region of the TCR) are presented to cognate anti-idiotypic CD4+ T cells, resulting in activation and proliferation of these cells. Interaction of idiotypic and anti-idiotypic T cells brought about by presentation of TCR idiopeptide may have important implications for T-cell vaccination and perpetuation of T-cell memory not requiring persisting antigen or long-lived memory cells.     

Isolation of Three Xylanase-Producing Strains of Actinomycetes and Their Identification Using Molecular Methods

Eighty-eight actinomycetes were isolated from 20 samples collected from different locations in and around Delhi, India. Among these, 69 isolates were found positive for xylanase production. Of 69 isolates, 3 (SN32, SN77, and SN83) produced >125 IU/ml xylanase. Modern genetic tools were used for revealing the identities of these potent xylanase producers. The selected isolates were categorized under the genus Streptomyces based on their cultural and morphologic characteristics. Genetic diversity among these species of Streptomyces was established based on restriction length fragment polymorphism and random amplified polymorphic DNA analysis. The closest phylogenetic neighbours according to the 16S rRNA gene-sequence data for the three isolates SN32, SN77, and SN83 were Streptomyces cyaneus, S. tendae, and S. caelestis, respectively.     

Dosimetric impact of Acuros XB on cervix radiotherapy using RapidArc technique: a dosimetric study

BackgroundAcuros XB (AXB) may predict better rectal toxicities and treatment outcomes in cervix carcinoma. The aim of the study was to quantify the potential impact of AXB computations on the cervix radiotherapy using the RapidArc (RA ) technique as compared to anisotropic analytical algorithm (AA) computations.Materials and methodsA cohort of 30 patients previously cared for cervix carcinoma (stages II–IIIB) was selected for the present analysis. The RA plans were computed using AA and AXB dose computation engines under identical beam setup and MLC pattern.ResultsThere was no significant (p > 0.05) difference in D_95% and D_98% to the planning target volume (PTV); moreover, a significant (p < 0.05) rise was noticed for mean dose to the PTV (0.26%), D_50% (0.26%), D_2% (0.80%) and V_110% (44.24%) for AXB computation as compared to AA computations. Further, AXB estimated a significantly (p < 0.05) lower value for maximum and minimum dose to the PTV. Additionally, there was a significant (p < 0.05) reduction observed in mean dose to organs at risk (OARs) for AXB computation as compared to AA, though the reduction in mean dose was non-significant (p > 0.05) for the rectum. The maximum difference observed was 4.78% for the rectum V_50Gy, 1.72%, 1.15% in mean dose and 2.22%, 1.48% in D_2% of the left femur and right femur, respectively, between AA and AXB dose estimations.ConclusionFor similar target coverage, there were significant differences observed between the AAA and AXB computations. AA underestimates the V_50Gy of the rectum and overestimates the mean dose and D_2% for femoral heads as compared to AXB. Therefore, the use of AXB in the case of cervix carcinoma may predict better rectal toxicities and treatment outcomes in cervix carcinoma using the RA technique.     

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.