Biophysics of malarial parasite exit from infected erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.     

Genetic and environmental effects on age at first farrowing in sows in southeastern Brazil

We evaluated genetic and environmental factors affecting age at first farrowing of sows in the Brazilian southeast. For this purpose, 466 observations regarding the age at first farrowing were made for Dalland-C40? animals belonging to two herds. The effects of the environmental factors on this trait were assessed by means of a model that included, as random effects, the influence of the sow's father and mother and, as fixed effects, the influence the year of birth, the herd and the birth season, along with the covariable litter size at birth. The variance components were estimated using the derivative-free restricted maximum likelihood method. The estimated mean was 354.8 ± 25.87 days, with a coefficient of variation of 7.29%. Significant effects on the trait were observed for the herd, the year and the season of birth; but a linear effect of litter size at birth on the age at first farrowing was not observed. The boar did not significantly contribute to the variation occurring among the sows, whereas the sow's mother caused significant variation. The heritability estimate for the age at first farrowing was 0.44 ± 0.15, which is considered high. We concluded that herd effect and year and season of birth should be taken into consideration for an accurate genetic comparison; consequently, the animals should be joined into contemporary groups.     

RET mutational spectrum in Hirschsprung disease: evaluation of 601 Chinese patients

Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5' untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.     

Testing Equality between Two Diagnostic Procedures in Paired‐Sample Ordinal Data

When a new diagnostic procedure is developed, it is important to assess whether the diagnostic accuracy of the new procedure is different from that of the standard procedure. For paired‐sample ordinal data, this paper develops two test statistics for testing equality of the diagnostic accuracy between two procedures without assuming any parametric models. One is derived on the basis of the probability of correctly identifying the case for a randomly selected pair of a case and a non‐case over all possible cutoff points, and the other is derived on the basis of the sensitivity and specificity directly. To illustrate the practical use of the proposed test procedures, this paper includes an example regarding the use of digitized and plain films for screening breast cancer. This paper also applies Monte Carlo simulation to evaluate the finite sample performance of the two statistics developed here and notes that they can perform well in a variety of situations. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects of precursors on serially propagated Digitalis lanata leaf and root cultures

Serially propagated Digitalis lanata leaf and root cultures established from germinated seeds were studied for digoxin production. Leaf cultures were grown and maintained in a medium containing benzyl adenine, and root cultures in the same medium with indoleacetic acid. A consistently high digoxin content, as determined by radio-immunoassay, is present in the leaf culture (9.0 mg % dry wt) and in root culture (1.9 mg % dry wt) as compared to unorganized cells (0.06 mg % dry wt). Leaf liquid cultures grew very rapidly as compared to the root cultures and the unorganized cell suspension cultures. The concentration of digoxin increased in both leaf and root cultures by adding to the medium either sodium glycocholate, cholesteryl acetate, or progesterone. Smilageninacetate increased the digoxin content of root cultures but not that of leaf cultures. Lanosterol and 5β-androstan-3,17-dione did not significantly increase the concentration of digoxin. Deoxycholic acid was toxic to the tissues studied.     

A simple test of the homogeneity of risk difference in sparse data: an application to a multicenter study

In this paper, we focus discussion on testing the homogeneity of risk difference for sparse data, in which we have few patients in each stratum, but a moderate or large number of strata. When the number of patients per treatment within strata is small (2 to 5 patients), none of test procedures proposed previously for testing the homogeneity of risk difference for sparse data can really perform well. On the basis of bootstrap methods, we develop a simple test procedure that can improve the power of the previous test procedures. Using Monte Carlo simulations, we demonstrate that the test procedure developed here can perform reasonable well with respect to Type I error even when the number of patients per stratum for each treatment is as small as two patients. We evaluate and study the power of the proposed test procedure in a variety of situations. We also include a comparison of the performance between the test statistics proposed elsewhere and the test procedure developed here. Finally, we briefly discuss the limitation of using the proposed test procedure. We use the data comparing two chemotherapy treatments in patients with multiple myeloma to illustrate the use of the proposed test procedure.     

Mitochondrial hydrogen peroxide production alters oxygen consumption in an oxygen-concentration-dependent manner

Metabolic responses of mammalian cells toward declining oxygen concentration are generally thought to occur when oxygen limits mitochondrial ATP production. However, at oxygen concentrations markedly above those limiting to mitochondria, several mammalian cell types display reduced rates of oxygen consumption without energy stress or compensatory increases in glycolytic ATP production. We used mammalian Jurkat T cells as a model system to identify mechanisms responsible for these changes in metabolic rate. Oxygen consumption was 31% greater at high oxygen (150–200 μM) compared to low oxygen (5–10 μM). Hydrogen peroxide was implicated in the response as catalase prevented the increase in oxygen consumption normally associated with high oxygen. Cell-derived hydrogen peroxide, predominately from the mitochondria, was elevated with high oxygen. Oxygen consumption related to intracellular calcium turnover was shown, through EDTA chelation and dantrolene antagonism of the ryanodine receptor, to account for 70% of the response. Oligomycin inhibition of oxygen consumption indicated that mitochondrial proton leak was also sensitive to changes in oxygen concentration. Our results point toward a mechanism in which changes in oxygen concentration influence the rate of hydrogen peroxide production by mitochondria, which, in turn, alters cellular ATP use associated with intracellular calcium turnover and energy wastage through mitochondrial proton leak.     

Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.): EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5′ and 3′ sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.     

黄芩的花粉母细胞减数分裂及核型分析

采用压片法,对黄芩花粉母细胞减数分裂及核型进行了研究。结果表明:黄芩的大多数花粉母细胞减数分裂中染色体的行为正常,在终变期同源染色体配对后可形成9个二价体,后期Ⅰ染色体以9∶9的方式向细胞两极分离,其减数分裂为同时型;在少数花粉母细胞减数分裂中观察到落后染色体、染色体桥等异常行为;其花粉粒育性为76.49%。黄芩的染色体数目为2n=2X=18,核型公式为K(2n)=2X=18=16m+2 sm,染色体相对长度组成为2n=1 s+4M1+3M2+1L,其核型为"1A"型。