Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses

Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5′-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses

Evolution of the Kangmar Dome, southern Tibet: Structural, petrologic, and thermochronologic constraints

Structural, thermobarometric, and thermochronologic investigations of the Kangmar Dome, southern Tibet, suggest that both extensional and contractional deformational histories are preserved within the dome. The dome is cored by an orthogneiss which is mantled by staurolite + kyanite zone metasedimentary rocks; metamorphic grade dies out up section and is defined by a series of concentric kyanite-in, staurolite-in, garnet-in, and chloritoid-in isograds. Three major deformational events, two older penetrative events and a younger doming event, are preserved. The oldest event, D1, resulted in approximately E-W trending tight to isoclinal folds of bedding with an associated moderately to steeply north dipping axial planar foliation, S1. The second event, D2, resulted in a high strain mylonitic foliation, S2, which defines the domal structure, and an associated approximately N-S trending stretching and mineral alignment lineation. Shear sense during formation of S2 varied from dominantly top S shear on the south dipping flank of the dome to top N shear on the north dipping flank. The central part of the dome exhibits either opposing shear sense indicators or symmetric fabrics. Microtextural relations indicate that peak metamorphism occurred post-D1 and pre- to early D2 deformation. Quantitative thermobarometry yields peak metamorphic conditions of ∼445°C and 370 MPa in garnet zone rocks, increasing to 625°C and 860 MPa in staurolite + kyanite zone rocks. Pressures and temperatures increase with depth and northward within a single structural horizon across the dome and the apparent gradient in pressure is ∼20% of the expected gradient, suggesting that the rocks were subvertically shortened after the pressure gradient was frozen in. Mica 40Ar/39Ar thermochronology yields 15.24 ± 0.05 to 10.94 ± 0.30 Ma cooling ages that increase with depth and young northward within a single structural horizon across the dome. Diffusion modeling of potassium feldspar 40Ar/39Ar spectra yield rapid cooling rates (∼10–30°C/Myr) between ∼11.5 and 10 Ma and apatite fission track ages range from 7.9 ± 3.0 to 4.1 ± 1.9 Ma, with a mean age of ∼5.5 Ma. Both data sets show symmetric cooling across the dome between ∼11 and 5.5 Ma. The S2 mylonitic foliation, peak metamorphic isobars and isotherms, and mica 40Ar/39Ar isochrons are domed, whereas potassium feldspar 40Ar/39Ar and apatite fission track isochrons are not, suggesting that doming occurred at ∼11 Ma. Our data do not support simple, end-member metamorphic core complex-type extension, diapirism, or duplex models for gneiss dome formation. Rather, we suggest that the formation of extensional fabrics occurred within a zone of coaxial strain in the root zone of the Southern Tibetan Detachment System (STDS), implying that normal slip along the STDS and extensional fabrics within the Kangmar Dome were the result of gravitational collapse of overthickened crust. Subsequent doming during the middle Miocene is attributed to thrusting upward and southward over a north dipping ramp above cold Tethyan sediments. Middle Miocene thrust faulting in the Kangmar Dome region is synchronous with continued normal slip along the STDS and thrust motion along the Renbu Zedong thrust fault, suggesting that extension and contraction was occurring simultaneously within southern Tibet

In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-μm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart

Sarcomere length dependent effects on the interaction between cTnC and cTnI in skinned papillary muscle strips

Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how the sarcomeres detect the length changes and convert them into altered activation of thin filament. In this study we investigated how the C-domain of cardiac troponin I (cTnI) functionally and structurally responds to the comprehensive effects of the Ca2+, crossbridge, and sarcomere length of chemically skinned myocardial preparations. Using our in situ technique which allows for simultaneous measurements of time-resolved FRET and mechanical force of the skinned myocardial preparations, we measured changes in the FRET distance between cTnI(167C) and cTnC(89C), labeled with FRET donor and acceptor, respectively, as a function of [Ca2+], crossbridge state and sarcomere length of the skinned muscle preparations. Our results show that [Ca2+], cross-bridge feedback and sarcomere length have different effects on the structural transition of the C-domain cTnI. In particular, the interplay between crossbridges and sarcomere length has significant impacts on the functional structural change of the C-domain of cTnI in the relaxed state. These novel observations suggest the importance of the C-domain of cTnI and the dynamic and complex interplay between various components of myofilament in the LDA mechanism. •The conformational change of C-domain of cTnI is Ca2+ and crossbridge states dependent.•The C-domain of cTnI is sensitive to sarcomere length and crossbridge states changes only in relaxed muscle.•The dynamic behavior of C-domain of cTnI is closely related to the binding states of crossbridges on the actin filament.•Changes in the structural dynamics of C-domain of cTnI reflect its functional importance in the thin filament regulation

Structural, Petrological and Thermal Evolution of a Tertiary Ductile Strike-Slip Shear Zone, Diancang Shan, Yunnan

International audienceThe Diancang Shan, a horst massif within the Red River fault zone in Yunnan, Peo-ple's Republic of China, preserves a structural, petrological, and thermal record of two distinct phases of tectonic activity: a left-lateral ductile shear that terminated between 20 and 17 Ma and a ductile-to-brittle phase of normal faulting which began at 4.7 Ma and remains active. Mylonitic rocks in the core of the range display an early, steep, high-temperature (HT), schistosity and a horizontal stretching lineation that are both parallel to the trend of the belt. Kinematic indicators indicate that shear was left-lateral. The complex shape of the HT schistosity at the southern termination of the massif likely results from a large-scale, oblique, left-lateral C' shear plane that dismembered the shear zone and separated the Ailao Shan and the Diancang Shan as left-lateral deformation terminated. Thermochronological and thermobarometric results suggest that the gneisses were partially unroofed during this event. Along the eastern edge of the Diancang Shan, the HT fabrics were overprinted by low-temperature structures during activation of east dipping normal faults. Cooling associated with this normal/right-lateral faulting along the Diancang Shan (and perhaps activation of the right-lateral/normal movement on the Range Front fault farther south along the Ailao Shan) began at 4.7:50.1 Ma. These results tend to support the view that extrusion of Indochina occurred along the left-lateral Red River shear zone between 35 and 19-17 Ma. Initiation of right-lateral/normal slip during the late Miocene may relate to eastward extensional collapse of the thickened Tibetan crust or, more probably, to initiation of the second phase of extrusion

A new fabrication technique to form complex polymethylmethacrylate microchannel for bioseparation

Recent studies show that reduction in cross-sectional area can be used to improve the concentration factor in microscale bioseparations. Due to simplicity in fabrication process, a step reduction in cross-sectional area is generally implemented in microchip to increase the concentration factor. But the sudden change in cross-sectional area can introduce significant band dispersion and distortion. This paper reports a new fabrication technique to form a gradual reduction in cross-sectional area in polymethylmethacrylate (PMMA) microchannel for both anionic and cationic isotachophoresis (ITP). The fabrication technique is based on hot embossing and surface modification assisted bonding method. Both one-dimensional and two-dimensional gradual reduction in cross-sectional area microchannels were formed on PMMA with high fidelity using proposed techniques. ITP experiments were conducted to separate and preconcentrate fluorescent proteins in these microchips. Thousand fold and ten thousand fold increase in concentrations were obtained when 10 × and 100 × gradual reduction in cross-sectional area microchannels were used for ITP