Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein

We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.     

Fluorometric detection of enzyme activity with synthetic supramolecular pores

The reversible blockage of synthetic pores formed by rigid-rod beta barrels, either by substrates or products, was used to sense a variety of enzymatic reactions in high-throughput format with "naked-eye" fluorescent detection. Improvement of sensor sensitivity beyond three orders of magnitude by straightforward internal mutations underscores the functional plasticity of rigid-rod beta barrels. Such detectors of enzyme activity with the aforementioned characteristics are needed in areas as diverse as proteomics and environmentally benign organic synthesis.     

Theaflavins depolymerize microtubule network through tubulin binding and cause apoptosis of cervical carcinoma HeLa cells

Here we studied the antiproliferative activity of theaflavins in cervical carcinoma HeLa cells by investigating their effects on cellular microtubules and purified goat brain tubulin. Theaflavins inhibited proliferation of HeLa cells with IC(50) value of 110 ± 2.1 μg/mL (p = < 0.01), caused cell cycle arrest at G(2)/M phase and induced apoptosis with alteration of expression of pro- and antiapoptotic proteins. Along with these antiproliferative activities, theaflavins act as microtubule depolymerizers. Theaflavins disrupted the microtubule network accompanied by alteration of cellular morphology and also decreased the polymeric tubulin mass of the cells. The polymerization of cold treated depolymerized microtubules in HeLa cells was prevented in the presence of theaflavins. In vitro polymerization of purified tubulin into microtubules was also inhibited by theaflavins with an IC(50) value of 78 ± 2.43 μg/mL (P < 0.01). Thus, disruption of cellular microtubule network of HeLa cells through microtubule depolymerization may be one of the possible mechanisms of antiproliferative activity of theaflavins.     

Development of Core Subset of Finger Millet Germplasm Using Geographical Origin and Data on 14 Quantitative Traits

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal food crop in Africa and South Asia. It is a hardy crop that can be grown in very diverse environments from almost at sea level to about 2400 m.a.s.l. Finger millet has an excellent food value as its seeds contain protein ranging from 7 to 14% and are particularly rich in methionine amino acid, iron, and calcium. Despite all these merits, this crop has been neglected from the main stream of crop improvement research. One of the means to boost its production and productivity is to enhance utilization of finger millet germplasm to breed superior varieties. Keeping this objective in view, a core subset of finger millet germplasm (622 accessions) based on origin and data on 14 quantitative traits was developed from the entire global collection of 5940 accessions held in the genebank at ICRISAT, Patancheru, India. The comparison of means, variances, frequency distribution, Shannon-Weaver diversity index (H′) and phenotypic correlations indicated that the core subset represents the entire collection. These tests indicated that sampling was optimal and the diversity has been captured very well in the core subset. The correlation analysis indicated that panicle exsertion and longest finger length could be given lower priority in the future germplasm evaluation work of finger millet.     

An efficient method for the purification and characterization of nematicidal azadirachtins A,B, and H,using MPLC and ESIMS

Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol-water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC(50) 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50-65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.     

Congenital cardiac defects in calves

In a 14-year study of calves with cardiac defects, 36 had 78 congenital cardiac defects: ectopia cordia cervicalis (n = 10 defects), common aortic trunk (n = 3 defects), dextraposed aorta (n = 8 defects), duplicated major trunks (n = 1 defect), hypoplastic aorta (n = 2 defects), interventricular septal defect (n = 11), interatrial septal defect (n = 2), left ventricular hypoplasia (n = 10), patent ductus arteriosus (n = 5), patent foramen ovale (n = 5), right ventricular hypoplasia (n = 10), cor triloculare biatriatum (n = 1), endocardial fibroelastosis with calcification (n = 3), and valvular hematomas (n = 7). All septal defects were high in location and ranged from 5 to 35 mm in diameter. One calf with a septal defect also had bilateral microphthalmia.     

Biological Control of Late Leaf Spot of Peanut (Arachis hypogaea) with Chitinolytic Bacteria

ABSTRACT Late leaf spot (LLS), caused by Phaeoisariopsis personata, is a foliar disease of groundnut or peanut (Arachis hypogaea) with high economic and global importance. Antifungal and chitinolytic Bacillus circulans GRS 243 and Serratia marcescens GPS 5, selected among a collection of 393 peanut-associated bacteria, were applied as a prophylactic foliar spray and tested for control of LLS. Chitin-supplemented application of B. circulans GRS 243 and S. marcescens GPS 5 resulted in improved biological control of LLS disease. Supplementation of bacterial cells with 1% (wt/vol) colloidal chitin reduced lesion frequency by 60% compared with application of bacterial cells alone, in the greenhouse. Chitinsupplemented application of GRS 243 and GPS 5 also resulted in improved and stable control of LLS in a repeated field experiment and increased the pod yields by 62 and 75%, respectively, compared with the control. Chitin-supplemented application of GPS 5 was tested in six onfarm trials, and the increase in pod yields was up to 48% in kharif (rainy season). A 55-kDa chitinase was purified from the cell-free culture filtrate of GPS 5 by affinity chromatography and gel filtration. Purified chitinase of S. marcescens GPS 5 (specific activity 120 units) inhibited the in vitro germination of P. personata conidia, lysed the conidia, and effectively controlled LLS in greenhouse tests, indicating the importance of chitinolysis in biological control of LLS disease by GPS 5.     

One-time transfers of cash or capital have long-lasting effects on microenterprises in Sri Lanka

Standard economic theory suggests that one-time business grants can have at most temporary effects, and accordingly, policies to increase incomes of the self-employed in developing countries typically rely on sustained engagement. In contrast, we found long-lasting impacts from one-time grants given in a randomized experiment to subsistence firms. Five years after we gave $100 or $200 to 115 of 197 male and 100 of 190 female Sri Lankan microenterprise owners, we found 10-percentage-point-higher enterprise survival rates, and $8-to-$12-per-month-higher profits for male-owned businesses that received the grants. Female-owned businesses showed no long-term (or short-term) impacts. Our follow-up investigation interviewed 94% of the original sample and collected survivorship data from the remaining 6%, demonstrating that tracking long-term outcomes is both feasible and worthwhile. The results suggest that one-off grants may have lasting impacts on some types of subsistence firms, challenging the view that sustained engagement is always required.