Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.     

Comparison of the gonadal response of wild and laboratory field voles (Microtus agrestis) to different photoperiods

Weanling male and female field voles from laboratory stock and from the F1 generation of wild-caught animals were placed in a long (16L:8D) or short (6L:18D) photoperiod for 28 or 56 days. Both types of field vole showed the well-established effect of photoperiod upon sexual maturation, with animals in the long photoperiod having larger and more active gonads than animals in the short photoperiod. After 28 and 56 days laboratory stock females were more mature, sexually, and had a higher growth rate than did Wild F1 females. There was no difference between the two types of males at 28 days, but by 56 days laboratory stock males were more sexually mature and had a higher growth rate than did Wild F1 males. These differences between the two types occurred in the long and short photoperiods. There was no interaction between photoperiod and type of vole. The use of laboratory stock animals in experiments could lead to an incorrect assessment of the effect of photoperiod in the control of seasonal breeding in wild populations.     

Fe2+-Induced Lysis and Lipid Peroxidation of Chromaffin Granules

Chromaffin granules, the catecholaminergic storage granules from adrenal chromaffin cells, lysed in 10(-9)-10(-7) M Fe2+. Lysis was accompanied by the production of malondialdehyde which results from lipid peroxidation. Both chromaffin granule lysis and malondialdehyde production were inhibited by the free radical trapping agent butylated hydroxytoluene but not by catalase and/or superoxide dismutase. The results suggest that lysis resulted from a direct transfer of electrons from Fe2+ to a component of the chromaffin granule membrane without the participation of either superoxide or hydrogen peroxide and may have resulted from lipid peroxidation. In some experiments, ascorbate alone induced chromaffin granule lysis which was inhibited by EDTA, EGTA, or deferoxamine. The lysis was probably caused by trace amounts of reducible polyvalent cation. Lysis sometimes occurred when Ca2+ was added with EGTA (10 microM free Ca2+ concentration) and was consistently observed together with malondialdehyde production in the presence of Ca2+, EGTA, and 10 microM Fe2+ (total concentration). The apparent Ca2+ dependency for chromaffin granule lysis and malondialdehyde production was probably caused by a trace reducible polyvalent ion displaced by Ca2+ from EGTA and not by a Ca2+-dependent reaction involving the chromaffin granule.     

The mechanical significance of the occlusal geometry of great ape molars in food breakdown

Analyses of dental function are an essential component of the study of human evolution. However, with few exceptions, they have utilized the traditional analogizing method of comparative anatomy, and have assumed rather than demonstrated that proposed adaptive characters confer a performance benefit. Since food reduction is a mechanical process, it is appropriate to measure performance using mechanical parameters, specifically the ability of a given morphology to induce failure in food particle by either of the two major regimes: crush and shear, corresponding to simple stresses (tensile and compressive) and shear stress, respectively. We apply finite elements stress analysis to model the relationship between the angulation of the intercuspal occlusal surfaces in a “puncture crushing” mode of mastication. On the basis of morphological data acquired from sectioned great ape molars, we have predicted the nature, magnitude and distribution of stress in a standard food particle by models representing each morphotype. Results indicate that the blunt-cusped molars ofHomo, the gradually-sloping supporting (buccal) cusps but high-angled guiding (lingual) cusps of the lower molars ofPan, and the high angled occlusal surfaces ofGorillaare all more likely to fracture small food particles by shear, while the gradually sloping occlusal surfaces ofPongomolars are more likely to break them down by “crush”. Mechanisms of food failure induced by molars ofPanandHomowill vary according to the orientation of the tooth–food contacting surfaces, which in turn will vary according to the size of the food particle. These genera may be able to break food down either by shear or by “crush”.     

Northern Ontario MDs seek solution to region's huge accidental-death toll.

A recently published study discussed the trauma-related death toll that plagues northeastern Ontario, which has a much higher death rate from nonintentional trauma than the rest of the province Dr. Gary Bota, one of the study authors hates calling these tragedies accidents because that implies a random, act-of-God nature that he does not accept. "They''re not accidents," he maintains. "They''re patterns, just like infectious diseases."     

Phylogeny of Selected Maxillopodan and Other Crustacean Taxa Based on 18S Ribosomal Nucleotide Sequences: A Preliminary Analysis

Relationships among the following taxa were examined based on 18S ribosomal RNA and DNA nucleotide sequences: Branchiopoda (Branchinecta packardi), Ostracoda (two podocopid species), Branchiura (Argulus nobilis), Pentastomida (Porocephalus crotali), Copepoda (Calanus pacificus, Macrocyclops albidus), Thoracica (Balanus eburneus, Calantica villosca), Acrothoracica (Trypetesa lampas), and Decapoda (Procambarus leonensis, Callinectes sapidus). Phylogenetic relationships were inferred by maximum parsimony and results evaluated by bootstrapping. With Branchinecta packardi as an outgroup, the following tree was inferred: the first branch leads to a node that joins a pentastome, a branchiuran and the two ostracodes; the second branch leads to a node that has the two copepods as a sister taxon to a clade that includes the acrothoracican and thoracicans; the last branch leads to a node joining the two decapods. The analyses suggest: (1) acrothoracicans diverged very early from the cirripede line and are not derived from a lepadomorph-like ancestor; (2) branchiurans are not related to either copepods or thecostracans but are closely allied with pentastomes; and (3) the Maxillopoda, broadly defined, is not a monophyletic taxon. These results differ from previous studies of these groups.     

Collagen concentrations in dissected tissue compartments of rat uterus on days 6, 7 and 8 of pregnancy.

Implantation and non-implantation sites were dissected into myometrial and stromal components; a decidual/embryonic region was obtained on Days 7 and 8 of pregnancy. The concentration of collagen (as a percentage of the dry weight of tissue), measured by hydroxyproline analysis, was significantly lower in the implantation regions than in the non-implant regions in all areas studied. The concentrations in the antimesometrial myometrium and stroma of the implantation region remained the same over the days studied. In contrast, the mesometrial collagen concentration in the implantation region declined from Day 6 to Day 8 of pregnancy. Collagen concentration was low within the decidual/embryonic tissue on Days 7 and 8 of pregnancy. Remodelling of collagen within the embryonic area appears to be an important feature of the uterine response to implantation in rats.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Assessment of the association between the frequency of micronucleus and p16INK4a/Ki-67 co-expression in patients with cervical intraepithelial lesions

Human papilloma virus (HPV) infection is the main etiological factor for cervical intraepithelial lesions (CIN). An important characteristic of this process is the loss of genome stability. Therefore, it is imperative to use biomarkers of DNA damage caused by genomic instability to identify high risk individuals. We investigated the frequency of micronuclei (MN) in peripheral blood lymphocytes (PBL) of 20 patients, diagnosed as histologically CIN 1 and 10 healthy controls. We also examined the frequency of other nuclear anomalies including nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in PBL of patients with CIN 1 and healthy controls, and evaluated the benefits of p16^INK4a and Ki-67 (p16^INK4a/Ki-67) immunohistochemical double staining for identifying cervical squamous cells that express HPV E6/E7 oncogenes. We analyzed the association between the frequency of MN in PBL and the amount of p16^INK4a/Ki-67 co-expression in CIN 1 patients to establish genomic instability. Among CIN 1 subjects, 15% exhibited diffuse p16^INK4a/Ki-67 co-expression and were considered high positive, 25% of the CIN 1 cases exhibited p16^INK4a/Ki-67 co-expression restricted to the lower part of the epithelium and were considered low positive and the remaining 60% of cases were negative. The frequency of MN, NPBs and NBUDs differed significantly among groups. We found a statistically significant positive correlation between p16^INK4a/Ki-67 co-expression and the frequency of MN, NPBs and NBUDs in PBL. Our findings demonstrate the efficacy of p16^INK4a/Ki-67 double immunostaining for histological samples with CIN 1. MN frequency in PBL might be useful for detecting genomic instability in cases of HPV infection and CIN.     

Rock geochemistry induces stress and starvation responses in the bacterial proteome

Interactions between microorganisms and rocks play an important role in Earth system processes. However, little is known about the molecular capabilities microorganisms require to live in rocky environments. Using a quantitative label‐free proteomics approach, we show that a model bacterium (Cupriavidus metallidurans CH34) can use volcanic rock to satisfy some elemental requirements, resulting in increased rates of cell division in both magnesium‐ and iron‐limited media. However, the rocks also introduced multiple new stresses via chemical changes associated with pH, elemental leaching and surface adsorption of nutrients that were reflected in the proteome. For example, the loss of bioavailable phosphorus was observed and resulted in the upregulation of diverse phosphate limitation proteins, which facilitate increase phosphate uptake and scavenging within the cell. Our results revealed that despite the provision of essential elements, rock chemistry drives complex metabolic reorganization within rock‐dwelling organisms, requiring tight regulation of cellular processes at the protein level. This study advances our ability to identify key microbial responses that enable life to persist in rock environments.