Segmentation of thin structures in electron micrographs using orientation fields

In this paper, we introduce a new approach for segmenting thin structures in electron micrographs. We introduce two new transforms, the Line Filter Transform (LFT) and the Orientation Filter Transform (OFT). The LFT can be viewed as an alternative to anisotropic diffusion algorithms that is particularly useful for thin structures. The OFT utilizes geometrical information about the structure by measuring correlations of local orientations in the image. By combining these methods with a contour extraction and labeling method we construct a segmentation method for thin structures in 2D images. We discuss how the method can be applied slice-by-slice to electron tomograms and illustrate the process by constructing two models of membrane structures from cellular tomograms. The suggested method has the advantage of being relatively insensitive to non-uniform contrast and high-contrast features such as ribosomes.     

Protection against malaria morbidity: Near-fixation of the α-thalassemia gene in a Nepalese population

We have previously reported that the Tharu people of the Terai region in southern Nepal have an incidence of malaria about sevenfold lower than that of synpatric non-Tharu people. In order to find out whether this marked resistance against malaria has a genetic basis, we have now determined in these populations the prevalence of candidate protective genes and have performed in-vitro cultures of Plasmodium falciparum in both Tharu and non-Tharu red cells. We have found significant but relatively low and variable frequencies of beta-thal, beta S, G6PD (-), and Duffy (a-b-) in different parts of the Terai region. The average in-vitro rate of invasion and of parasite multiplication did not differ significantly in red cells from Tharus versus those from non-Tharu controls. By contrast, the frequency of alpha-thalassemia is uniformly high in Tharus, with the majority of them having the homozygous alpha-/alpha-genotype and an overall alpha-thal gene (alpha-) frequency of .8. We suggest that holoendemic malaria has caused preferential survival of subjects with alpha-thal and that this genetic factor has enabled the Tharus as a population to survive for centuries in a malaria-holoendemic area. From our data we estimate that the alpha-thal homozygous state decreases morbidity from malaria by about 10-fold. This is an example of selection evolution toward fixation of an otherwise abnormal gene.     

STUDIES ON THE MECHANISM OF ACTION OF PEDERINE

Pederine, a drug extracted from the coleopter Paederus fuscipes, inhibits the growth of in vitro cultured cell lines at concentrations of the order of 1.5 nanogram/ml. Cytological examination shows a generalized cytotoxic effect. Analysis of macromolecular syntheses by the use of radioactive precursors shows that pederine causes an almost immediate block of protein and DNA synthesis, without affecting RNA synthesis. The effects on the synthesis of the two types of macromolecules remain nearly simultaneous even at the lowest active concentrations of pederine. Studies with cell-free systems show that the drug inhibits protein synthesis, whereas it is ineffective on the DNA-polymerizing activity. It seems, therefore, that the drug acts primarily on the amino acid-polymerizing system, and that the effect on DNA is secondary. This idea is strengthened by the observation that puromycin, a specific inhibitor of protein synthesis, also affects promptly DNA synthesis of in vitro cultured cells. Other authors have shown the same phenomenon with a number of inhibitors of protein synthesis; the properties of pederine support, therefore, the view that continuous protein synthesis is necessary for the maintenance of DNA replication in higher organisms.     

SecA Localization and SecA-Dependent Secretion Occurs at New Division Septa in Group B Streptococcus

Exported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted into the extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. These proteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containing the so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, and LPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown that presence of the YSIRKG/S motif (literally YSIRK_X3Gx₂S) in the signal peptide (SP) constitutes the targeting information for secretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied in GBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonical YSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did not alter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoring machineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate (CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatment indicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles. We conclude that the ±YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae.     

Genetic studies on the Tharu population of Nepal: restriction endonuclease polymorphisms of mitochondrial DNA.

The mitochondrial DNAs (mtDNAs) of 91 Tharus from Nepal were screened for restriction fragment length polymorphisms (RFLPs) using six highly informative restriction endonucleases. One pattern (morph) was found for BamHI, two for HpaI and HincII, three for HaeII, four for AvaII, and six for MspI. Two of the AvaII and four of the MspI morphs were "new" (not previously described). Virtually all of the "old" morphs found in the Tharus were previously observed in Orientals. The Oriental HaeII morph (HaeII-5) previously observed at a frequency of 5% was present in 25% of the Tharus. Of the 13 Tharu mtDNA types (defined by the six restriction endonuclease morphs) observed, five had previously been described ("old" types), all in Orientals. Three of these were unique for Orientals. All of the remaining eight "new" Tharu mtDNAs were all closely related to Oriental mtDNAs. Two of the "old" Tharu mtDNA types included the HpaI/HincII morph 1, a morph possibly indicative of the earliest human mtDNA types. From these data we have concluded that the Tharu mtDNAs are closely related to those of other Oriental populations. Further, our data support the hypothesis that human mtDNAs radiated from Asia.     

Use of reconstituted basal membranes for the study of invasion of human tumor cells: current status and future prospects

Tumor metastasis is the major cause of death of oncology patients. One of the characteristic properties acquired by the metastatic cell is the ability to cross basement membranes. These are compartments of extracellular matrix composed largely by collagen type IV, laminin and a heparan sulphate proteoglycan. Here we review the use of a reconstituted basement membrane (Matrigel) in the Boyden chamber assay (Chemoinvasion Assay) for the assessment of the invasiveness of tumor cells of human origin. The possibility of using this test for the rapid evaluation of human tumor specimens from operated patients is discussed.     

The GBS PI-2a pilus is required for virulence in mice neonates

Background

Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone.

Methodology/Principal Findings

In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity.

Conclusions/Significance

These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.