Folate binding and transport by rat kidney brush-border membrane vesicles

[3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with Km values of 6.7.10(-7) and 11.2.10(-7) M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Dynamic quenching studies of fluorophore-labelled myosin subfragment 1 and its alkali light chain subunits

The technique of fluorescence quenching by the non-ionic quenchers acrylamide and nicotinamide has been used to probe the accessibility of the environmentally sensitive N-(bromoacetyl)-N'-(1-sulpho-5-naphthyl) ethylenediamine (1,5-Br-AEDANS) fluorophore attached to either Cys-177 of the A1-light chain or the SH1 thiol (Cys-707) of the myosin subfragment (S1) heavy chain. Neither quencher caused any detrimental effects to the ATPase activities of S1 under the conditions of the experiments. It was found that the fluorophore on the isolated light chain was highly exposed to solvent and although this exposure was reduced on hybridization into S1(A1-AEDANS), the probe was still accessible to solvent. This exposure was unaltered by formation of binary complexes with either Mg.ATP or actin or by the formation of a weakly associated acto-S1 complex (in which the Cys-697 and Cys-707 residues of S1 were crosslinked with p-phenylenedimaleimide). The lack of corresponding change in lambda max of emission and quantum yield supported the quenching date and indicated that actin neither binds directly to this region nor induces any significant conformational changes in this locality despite the observation that the A1-Cys-707 moves some 3 nm closer to a point on actin in the weak-binding state (Trayer, H.R. and Trayer, I.P. (1988) Biochemistry, 27, 5718-5727). Parallel experiments with the fluorophore attached to the Cys-707 of the S1 indicated that this region was less accessible to solvent than the light chain thiol despite its ease of labelling. This exposure was not significantly altered by binary complex formation with actin and Mg.ATP, although spectral changes in the absence of quencher support the notion that some conformational change is occurring in this region.     

A novel type of bicoumarin rhamnoside from Lasiosipho eriocephalus

A new furanobicoumarin rhamnoside has been characterized from the whole plant extract of Lasiosiphon eriocephalus.     

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Controlling the Three-Dimensional Printing Mechanical Properties of Nostoc Sphaeroides System

The main purpose of this paper is to explore the opportunities for fresh Nostoc sphaeroides (N. sphaeroides) to be applied to 3D food printing. N. sphaeroides is rich in nutrients and its paste possesses shear thinning properties. It was found the product obtained by 3D food printing with fresh N. sphaeroides had poor printability and was easy to collapse. In this study, we compared the addition of different potato starch (2%, 4%, 6% and 8%) to the characteristics of 3D printing of the N. sphaeroides gel system. The results obtained from the rheological analysis showed that the 6% potato starch added to of N. sphaeroides gel can be utilized for 3D food printing. The addition of potato starch increased the viscosity of the mixture so the printed lines were not easily broken, and the “self-supporting ability” of the material itself was enhanced to maintain a good shape without collapse. Texture profile analysis also showed that the 6% starch added printed product had the best gumminess parameter. In order to get a better printed product, the effects of printing parameters (nozzle diameter (Dn), extrusion rate (Vd) and nozzle moving speed (Vn)) on material printing performance and product formability was tested. When Dn, Vd, Vn were = 1.2 mm, 20 mm³/s, 25 mm/s, respectively, the printed product was having similar to the target product, with less breakage and less the changing of shape. Overall results show that 3D printing technology is a rising method for producing N. sphaeroides-based new products.

     

Cost Effectiveness of Implementing Integrated Management of Neonatal and Childhood Illnesses Program in District Faridabad,India

Introduction

Despite the evidence for preventing childhood morbidity and mortality, financial resources are cited as a constraint for Governments to scale up the key health interventions in some countries. We evaluate the cost effectiveness of implementing IMNCI program in India from a health system and societal perspective.

Methods

We parameterized a decision analytic model to assess incremental cost effectiveness of IMNCI program as against routine child health services for infant population at district level in India. Using a 15-years time horizon from 2007 to 2022, we populated the model using data on costs and effects as found from a cluster-randomized trial to assess effectiveness of IMNCI program in Haryana state. Effectiveness was estimated as reduction in infant illness episodes, deaths and disability adjusted life years (DALY). Incremental cost per DALY averted was used to estimate cost effectiveness of IMNCI. Future costs and effects were discounted at a rate of 3%. Probabilistic sensitivity analysis was undertaken to estimate the probability of IMNCI to be cost effective at varying willingness to pay thresholds.

Results

Implementation of IMNCI results in a cumulative reduction of 57384 illness episodes, 2369 deaths and 76158 DALYs among infants at district level from 2007 to 2022. Overall, from a health system perspective, IMNCI program incurs an incremental cost of USD 34.5 (INR 1554) per DALY averted, USD 34.5 (INR 1554) per life year gained, USD 1110 (INR 49963) per infant death averted. There is 90% probability for ICER to be cost effective at INR 2300 willingness to pay, which is 5.5% of India’s GDP per capita. From a societal perspective, IMNCI program incurs an additional cost of USD 24.1 (INR 1082) per DALY averted, USD 773 (INR 34799) per infant death averted and USD 26.3 (INR 1183) per illness averted in during infancy.

Conclusion

IMNCI program in Indian context is very cost effective and should be scaled-up as a major child survival strategy.     

The fatty acid chain elongase,Elovl1, is required for kidney and swim bladder development during zebrafish embryogenesis

Very long chain fatty acids are required for sphingolipid synthesis, lipid homeostasis, myelin formation, epidermal permeability, and retinal function. Seven different enzymes are known to be involved in the elongation cycle of fatty acids, with different chain-length specificities. Elovl1 is one of those enzymes whose function has been linked mainly to the synthesis of sphingolipids and the epidermal barrier. However, the role of Elovl1 in organogenesis is not clear. In zebrafish, 2 Elovl1 genes, elovl1a and elovl1b, are highly expressed in the swim bladder, and elovl1b is also expressed in the kidney. We found that both elovl1 knockdown embryos contain increased levels of long chain fatty acids from carbon number 14 to 20 as compared to control embryos. Oil-Red-O staining shows that yolk lipid consumption is greatly reduced, whereas lipid droplets accumulate within the swim bladder. Notably, knockdown of either elovl1a or elovl1b affects the expression of genes involved in swim bladder development and impairs inflation of the swim bladder. Consistent with its expression in the pronephros, knockdown of elovl1b alone affects the expression of genes required for kidney development and reduces renal clearance. Our findings strongly suggest that both elovl1 genes are a key determinant of swim bladder and kidney development in zebrafish, which may be comparatively applicable to lung and kidney development in humans.     

Fine‐scale ecological and anthropogenic variables predict the habitat use and detectability of sloth bears in the Churia habitat of east Nepal

Once widespread throughout the tropical forests of the Indian Subcontinent, the sloth bears have suffered a rapid range collapse and local extirpations in the recent decades. A significant portion of their current distribution range is situated outside of the protected areas (PAs). These unprotected sloth bear populations are under tremendous human pressures, but little is known about the patterns and determinants of their occurrence in most of these regions. The situation is more prevalent in Nepal where virtually no systematic information is available for sloth bears living outside of the PAs. We undertook a spatially replicated sign survey‐based single‐season occupancy study intending to overcome this information gap for the sloth bear populations residing in the Trijuga forest of southeast Nepal. Sloth bear sign detection histories and field‐based covariates data were collected between 2 October and 3 December 2020 at the 74 randomly chosen 4‐km² grid cells. From our results, the model‐averaged site use probability (ψ ± SE) was estimated to be 0.432 ± 0.039, which is a 13% increase from the naïve estimate (0.297) not accounting for imperfect detections of sloth bear signs. The presence of termite mound and the distance to the nearest water source were the most important variables affecting the habitat use probability of sloth bears. The average site‐level detectability (p ± SE) of sloth bear signs was estimated to be 0.195 ± 0.003 and was significantly determined by the index of human disturbances. We recommend considering the importance of fine‐scale ecological and anthropogenic factors in predicting the sloth bear‐habitat relationships across their range in the Churia habitat of Nepal, and more specifically in the unprotected areas.     

Isolation of RNA from apple skin

A method has been developed for the isolation of RNA from apple skin. The method involves an adaptation of the Manning (1991) method and includes a high-salt extraction step and a final purification step through a CsCl cushion. The RNA isolated was of high quality and produced good hybridization signals in northern blot analyses.