Restriction endonuclease analysis of the transducing bacteriophage lambda rif d18

The rif d18 bacteriophage carries essential parts of the E. coli genome which can not be mapped by conventional methods. The phage DNA was analysed with four restriction endonucleases (endo R. BamHI, Sall, HpaI and EcoRI) and a physical map was constructed.     

Removing the threat of diclofenac to critically endangered Asian vultures

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genusGyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captiveGyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vultureGyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered AsianGyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40G. africanus. Subsequently, sixG. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species(Gyps bengalensis,Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity toGyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India.     

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

Key message

Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard.

Abstract

Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (N_o without added N and N₁₀₀ added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

     

Novel intron length polymorphic (ILP) markers from starch biosynthesis genes reveal genetic relationships in Indian wheat varieties and related species

Molecular Biology Reports - Introns experience lesser selection pressure, thus are liable for higher polymorphism. Intron Length Polymorphic (ILP) markers designed from exon-flanking introns...     

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.     

A novel concept of radiosynthesis of a 99mTc-labeled dimeric RGD peptide as a potential radiotracer for tumor imaging

Radiolabeled Arg-Gly-Asp (RGD) peptides are promising agents for non invasive imaging of α_vβ₃ expression in malignant tumors. The integrin α_vβ₃ binding affinity and consequent tumor uptake could be improved when a dimeric RGD peptide is used as the targeting moiety instead of a monomer. Towards this, a novel approach was envisaged to synthesize a ^99mTc labeled dimeric RGD derivative using a RGD monomer and [^99mTcN]⁺² intermediate. The dithiocarbamate derivative of cyclic RGD peptide G₃-c(RGDfK) (G₃ = Gly-Gly-Gly, f = Phe, K = Lys) was synthesized and radiolabeled with [^99mTcN]⁺² intermediate to form the ^99mTcN-[G₃-c(RGDfK)]₂ complex in high yield (～98%). Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed good tumor uptake [4.61 ± 0.04% IA/g at 30 min post-injection] with fast clearance of the activity from non-target organs/tissue. Scintigraphic imaging studies showed visible accumulation of activity in the tumor with appreciable target to background ratio.     

Precise periodic components estimation for chronobiological signals through Bayesian Inference with sparsity enforcing prior

The toxicity and efficacy of more than 30 anticancer agents present very high variations, depending on the dosing time. Therefore, the biologists studying the circadian rhythm require a very precise method for estimating the periodic component (PC) vector of chronobiological signals. Moreover, in recent developments, not only the dominant period or the PC vector present a crucial interest but also their stability or variability. In cancer treatment experiments, the recorded signals corresponding to different phases of treatment are short, from 7 days for the synchronization segment to 2 or 3 days for the after-treatment segment. When studying the stability of the dominant period, we have to consider very short length signals relative to the prior knowledge of the dominant period, placed in the circadian domain. The classical approaches, based on Fourier transform (FT) methods are inefficient (i.e., lack of precision) considering the particularities of the data (i.e., the short length). Another particularity of the signals considered in such experiments is the level of noise: such signals are very noisy and establishing the periodic components that are associated with the biological phenomena and distinguishing them from the ones associated with the noise are difficult tasks. In this paper, we propose a new method for the estimation of the PC vector of biomedical signals, using the biological prior informations and considering a model that accounts for the noise. The experiments developed in cancer treatment context are recording signals expressing a limited number of periods. This is a prior information that can be translated as the sparsity of the PC vector. The proposed method considers the PC vector estimation as an Inverse Problem (IP) using the general Bayesian inference in order to infer the unknown of our model, i.e. the PC vector but also the hyperparameters (i.e the variances). The sparsity prior information is modeled using a sparsity enforcing prior law. In this paper, we propose a Student’s t distribution, viewed as the marginal distribution of a bivariate normal-inverse gamma distribution. We build a general infinite Gaussian scale mixture (IGSM) hierarchical model where we assign prior distributions also for the hyperparameters. The expression of the joint posterior law of the unknown PC vector and hyperparameters is obtained via Bayes rule, and then, the unknowns are estimated via joint maximum a posteriori (JMAP) or posterior mean (PM). For the PM estimator, the expression of the posterior distribution is approximated by a separable one, via variational Bayesian approximation (VBA), using the Kullback-Leibler (KL) divergence. For the PM estimation, two possibilities are considered: an approximation with a partially separable distribution and an approximation with a fully separable one. Both resulting algorithms corresponding to the PM estimation and the one corresponding to the JMAP estimation are iterative algorithms. The algorithms are presented in detail and are compared with the ones corresponding to the Gaussian model. We examine the convergency of the algorithms and give simulation results to compare their performances. Finally, we show simulation results on synthetic and real data in cancer treatment applications. The real data considered in this paper examines the rest-activity patterns of KI/KI Per2::luc mouse, aged 10 weeks, singly housed in RealTime Biolumicorder (RT-BIO).     

Involuntary hospitalizations in the psychiatric hospital Jankomir before and following the alterations and amendments made to ZZODS

Schizophrenia and other psychotic disorders as well as the delirium caused by abstinence from alcohol and accute state of drunkenness appear at the very top of the list of factors, which are positively correlated with involuntary hospitalization of patients. This is at the same time a confirmation of the data found in literature considering psychosis an essential factor of involuntary hospitalization; the same referring to the male sex was not, however, confirmed by the results obtained in the first and second research period. Regarding the positive correlation between schizophrenia and other psychotic disturbances, dementia, delirium and other cognitive impairments including the delirium caused by abstinence from alcohol and an accute state of drunkenness on the one side and the high rate of involuntary hospitalization on the other, there is no statistically significant difference between the period preceding and the period following the alterations and amendments to the Law on the protection of patients with mental disorders.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.