Science and Technology

Development of amphibolite-facies transposition fabrics in the northern Canadian Cordilleran hinterland occurred diachronously in the Permian–Triassic, Early Jurassic, Middle Jurassic to Early Cretaceous, and Early to mid-Cretaceous. Rocks tectonized in the Permian–Trias- sic and Early Jurassic were exhumed in the Early Jurassic, while rocks immediately to the northeast (toward the foreland) were not buried and heated until the Middle Jurassic to mid-Cretaceous. Early Jurassic to mid-Cretaceous emplacement of the Yukon-Tanana terrane on the North American continental margin, together with the imbrication of parautochthonous rocks, formed a foreland-propagating orogenic wedge. Cooler rocks in front of the wedge were progressively buried and metamorphosed to amphibolite facies from the Jurassic to mid- Cretaceous as they were underthrust into a spatially and temporally transient distributed ductile shear zone near the base of the overrid- ing wedge. Rocks previously incorporated into this zone were displaced upward and exhumed through the combined effects of renewed underplating at depth and compensating extensional and erosional denudation above to maintain a critically tapered wedge. Extensional exhumation of the metamorphic hinterland in the mid-Cretaceous marked the collapse and end of orogen-perpendicular wedge dynam- ics in operation since the Early Jurassic. Rocks incorporated into the midcrustal shear zone in the Middle Jurassic to mid-Cretaceous were exhumed in the mid-Cretaceous along southeast-directed (orogen-parallel) extensional faults from beneath a supracrustal “lid” tectonized in the Permian–Triassic and Early Jurassic. Like the Himalayan orogen and eastern Alps, orogen-parallel extension developed in an orthogo- nal plate-convergent setting, simultaneous with, and bounded by, orogen-parallel strike-slip faulting that facilitated northwestward lateral extrusion of rocks normal to the direction of convergence.

Sierra Negra volcano, the most voluminous shield volcano in the Galápagos archipelago and one of the largest basaltic calderas in the world, erupted on October 22, 2005 after more than 25years of quiescence. GPS and satellite radar interferometry (InSAR) monitoring of the deformation of the caldera floor in the months prior to the eruption documented extraordinary inflation rates (1cm/day). The total amount of uplift recorded since monitoring began in 1992 approached 5m at the center of the caldera over the eight days of the eruption the caldera floor deflated a maximum of 5 m and subsquently renewed its inflation, but at a decelerating rate. To gain insight into the nature of the subsurface mass/density changes associated with the deformation, gravity measurements performed in 2005, 2006, and 2007 are compared to previous measurements from 2001-2002 when the volcano underwent a period of minor deflation and magma withdrawal. The residual gravity decrease between 2001-2002 and 2005 is among the largest ever recorded at an active volcano (−950 μGal) and suggests that inflation was accompanied by a relative density decrease in the magmatic system. Forward modeling of the residual gravity data in 4D (from 2002 to 2005) gives an estimate of the amount of vesiculation in the shallow sill required to explain the observed gravity variations. Geochemical constraints from melt inclusion and satellite remote-sensing data allow us to estimate the pre-eruptive gas content of the magma and place constraints on the thickness of the gas-rich sill necessary to produce the gravity anomalies observed. Results suggest that reasonable sill thicknesses (700–800m) and bubble contents (10–50 volume %) can explain the large decrease in residual gravity prior to eruption. Following the eruption (2006 and 2007), the deformation and gravity patterns suggest re-equilibration of the pressure regime in the shallow magma system via a renewed influx of relatively gas-poor magma into the shallow parts of the system.

A rapid system of sampling for strawberry aphids, Chaetosiphon fragaefolii (Cockerell) was developed for use by pest management scouts. Regression equations relating mean numbers of aphids/leaf, variances of those means and the proportion of unifested leaves (Po) were developed from samples of aphids from single leaves. Using the equations, mean aphid density per leaf and standard errors can be estimated from Po and the sample size. The accuracy of the estimations were tested on data from 155 samples from commercial strawberry fields sampled by a professional pest management company. Means estimated from Po were sufficiently accurate for the intended purpose and only 2 hours were required to sample 300 leaves compared to 16 hours when all aphids on all leaves were counted from only 80 leaves. An electronic recorder was programmed to prompt an IPM scout for data entry, allow correction of errors and permit sampling from different subplots within a field.

Alternatively activated macrophages are critical in host defense against parasites and are protective in inflammatory bowel disease, but contribute to pathology in asthma and solid tumors. The mechanisms underlying alternative activation of macrophages are only partially understood and little is known about their amenability to manipulation in pathophysiological conditions. Herein, we demonstrate that Src homology 2-domain-containing inositol-5'-phosphatase (SHIP)-deficient murine macrophages are more sensitive to IL-4-mediated skewing to an alternatively activated phenotype. Moreover, SHIP levels are decreased in macrophages treated with IL-4 and in murine GM-CSF-derived and tumor-associated macrophages. Loss of SHIP and induction of alternatively activated macrophage markers, Ym1 and arginase I (argI), were dependent on phosphatidylinositol 3-kinase (PI3K) activity and argI induction was dependent on the class IA PI3Kp110δ isoform. STAT6 was required to reduce SHIP protein levels, but reduced SHIP levels did not increase STAT6 phosphorylation. STAT6 transcription was inhibited by PI3K inhibitors and enhanced when SHIP was reduced using siRNA. Importantly, reducing SHIP levels enhanced, whereas SHIP overexpression or blocking SHIP degradation reduced, IL-4-induced argI activity. These findings identify SHIP and the PI3K pathway as critical regulators of alternative macrophage activation and SHIP as a target for manipulation in diseases where macrophage phenotype contributes to pathology.

Background & Aims Crohn’s disease (CD) is associated with a dysregulated immune response to commensal micro-organisms in the intestine. Mice deficient in inositol polyphosphate 5′-phosphatase D (INPP5D, also known as SHIP) develop intestinal inflammation resembling that of patients with CD. SHIP is a negative regulator of PI3Kp110α activity. We investigated mechanisms of intestinal inflammation in Inpp5d−/− mice (SHIP-null mice), and SHIP levels and activity in intestinal tissues of subjects with CD. <p>Methods We collected intestines from SHIP-null mice, as well as Inpp5d+/+ mice (controls), and measured levels of cytokines of the interleukin 1 (IL1) family (IL1α, IL1β, IL1ra, and IL6) by enzyme-linked immunosorbent assay. Macrophages were isolated from lamina propria cells of mice, IL1β production was measured, and mechanisms of increased IL1β production were investigated. Macrophages were incubated with pan−phosphatidylinositol 3-kinase inhibitors or PI3Kp110α-specific inhibitors. Some mice were given an antagonist of the IL1 receptor; macrophages were depleted from ilea of mice using clodronate-containing liposomes. We obtained ileal biopsies from sites of inflammation and peripheral blood mononuclear cells (PBMCs) from treatment-naïve subjects with CD or without CD (controls), and measured SHIP levels and activity. PBMCs were incubated with lipopolysaccharide and adenosine triphosphate, and levels of IL1β production were measured. <p>Results Inflamed intestinal tissues and intestinal macrophages from SHIP-null mice produced higher levels of IL1B and IL18 than intestinal tissues from control mice. We found PI3Kp110α to be required for macrophage transcription of Il1b. Macrophage depletion or injection of an IL1 receptor antagonist reduced ileal inflammation in SHIP-null mice. Inflamed ileal tissues and PBMCs from patients with CD had lower levels of SHIP protein than controls (P < .0001 and P < .0002, respectively). There was an inverse correlation between levels of SHIP activity in PBMCs and induction of IL1β production by lipopolysaccharide and adenosine triphosphate (R2 = .88). <p>Conclusions Macrophages from SHIP-deficient mice have increased PI3Kp110α-mediated transcription of Il1b, which contributes to spontaneous ileal inflammation. SHIP levels and activity are lower in intestinal tissues and peripheral blood samples from patients with CD than controls. There is an inverse correlation between SHIP activity and induction of IL1β production by lipopolysaccharide and adenosine triphosphate in PBMCs. Strategies to reduce IL1B might be developed to treat patients with CD found to have low SHIP activity.

Type IIB fast fibres are typically demonstrated in human skeletal muscle by histochemical staining for the ATPase activity of myosin heavy-chain (MyHC) isoforms. However, the monoclonal antibody specific for the mammalian IIB isoform does not detect MyHC IIB protein in man and MyHC IIX RNA is found in histochemically identified IIB fibres, suggesting that the IIB protein isoform may not be present in man; if this is not so, jaw-closing muscles, which express a diversity of isoforms, are likely candidates for their presence. ATPase histochemistry, immunohistochemistry polyacrylamide gel electrophoresis and in situ hybridization, which included a MyHC IIB-specific mRNA riboprobe, were used to compare the composition and RNA expression of MyHC isoforms in a human jaw-closing muscle, the masseter, an upper limb muscle, the triceps, an abdominal muscle, the external oblique, and a lower limb muscle, the gastrocnemius. The external oblique contained a mixture of histochemically defined type I, IIA and IIB fibres distributed in a mosaic pattern, while the triceps and gastrocnemius contained only type I and IIA fibres. Typical of limb muscle fibres, the MyHC I-specific mRNA probes hybridized with histochemically defined type I fibres, the IIA-specific probes with type IIA fibres and the IIX-specific probes with type IIB fibres. The MyHC IIB mRNA probe hybridized only with a few histochemically defined type I fibres in the sample from the external oblique; in addition to this IIB message, these fibres also expressed RNAs for MyHC I, IIA and IIX. MyHC IIB RNA was abundantly expressed in histochemical and immunohistochemical type IIA fibres of the masseter, together with transcripts for IIA and in some cases IIX. No MyHC IIB protein was detected in fibres and extracts of either the external oblique or masseter by immunohistochemistry, immunoblotting and electrophoresis. Thus, IIB RNA, but not protein, was found in the fibres of two different human skeletal muscles. It is believed this is the first report of the substantial expression of IIB mRNA in man as demonstrated in a subset of masseter fibres, but rarely in limb muscle, and in only a few fibres of the external oblique. These findings provide further evidence for the complexity of myosin gene expression, especially in jaw-closing muscles.

We report a single step, simple, repeatable, rapid and reliable technique for simultaneous immunocytochemical staining with two or more rabbit polyclonal antibodies. This technique, which we have dubbed the ‘Pretty Poly’ method, is based on conjugating the antibodies with commercially available, fluorophore-tagged Staphylococcal protein-A (SP-A). Staining is illustrated at the calyx type presynaptic nerve terminal of the chick ciliary ganglion with antibodies directed against three nerve terminal proteins: neurofilaments of the axonal cytoskeleton, and two secretory vesicle proteins, SV2 and cysteine string protein (CSP). Images were deblurred with an iterative deconvolution protocol. Staining with a single polyclonal antibody was bright and had a resolution approaching light microscope limit. Treatment with two different polyclonal antibodies conjugated with contrasting dye-tagged protein-A resulted in double staining without significant crossover that was fully equivalent to the standard primary/secondary technique. The same single step protocol was used to stain with all three rabbit polyclonal antibodies or to combine the technique with a standard monoclonal primary/secondary antibody stain. Thus, the Pretty Poly protocol is a highly flexible, simple and yet effective staining technique that essentially solves the problem of co-staining with multiple polyclonal rabbit antibodies.

Presynaptic CaV2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive GαO G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of CaV2.2 with syntaxin 1 and GαO. CaV2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with CaV2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the CaV2.2 puncta, whereas the bulk colocalized with MUNC18-1. GαO, whether in its trimeric or monomeric state, did not coimmunoprecipitate with CaV2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with CaV2.2 and GαO, as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.

A one-tube nested polymerase chain reaction (PCR) method for the diagnosis of paucibacillary leprosy was developed using the repetitive RLEP sequence as a target. Detection of the PCR products was simplified by the adaptation of a colorimetric method. The test was specific for Mycobacterium leprae, and the sensitivity of the assay was 1 fg of purified genomic M. leprae DNA (less than one genome). Complete concordance was seen between the development of color and resolution on agarose gels. The results of frozen skin sections from untreated patients showed that the assay could detect 100% of multibacillary samples [bacterial index (BI) of 2 or more] and 69% and 70% of the samples with Bis of 1 and 0, respectively. The use of one-tube nested PCR in assessing the effectiveness of multidrug therapy (MDT) in leprosy also was determined. The simplified colorimetric assay was found to be sensitive, rapid and specific, and is suitable for use in routing diagnostic laboratories.

Mcl-1 (myeloid cell leukaemia-1) is a Bcl-2 family member with short-term pro-survival functions but whose other functions, demonstrated by embryonic lethality of knockout mice, do not involve apoptosis. In the present study, we show a cell-cycle-regulatory role of Mcl-1 involving a shortened form of the Mcl-1 polypeptide, primarily localized to the nucleus, which we call snMcl-1. snMcl-1 interacts with the cell-cycle-regulatory protein Cdk1 (cyclin-dependent kinase 1; also known as cdc2) in the nucleus, and Cdk1 bound to snMcl-1 was found to have a lower kinase activity. The interaction with Cdk1 occurs in the absence of its cyclin partners and is enhanced on treatment of cells with G2/M blocking agents, but not by G1/S blocking. The snMcl-1 polypeptide is present during S and G2 phases and is negligible in G1. Overexpression of human Mcl-1 in a murine myeloid progenitor cell line resulted in a lower rate of proliferation. Furthermore, Mcl-1-overexpressing cells had lower total Cdk1 kinase activity compared with parental cells, in both anti-Cdk1 and anti-cyclin B1 immunoprecipitates. The latter results suggest that binding to snMcl-1 alters the ability of Cdk1 to bind its conventional partner, cyclin B1. Given the important role of Cdk1 in progression through G2 and M phases, it is probable that the inhibition of Cdk1 activity accounts for the inhibitory effect of Mcl-1 on cell growth.