Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce ...cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.
Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist, 11-cis ...retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and nonvisual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here, we show that a nonnatural retinal analog, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as a JSR1 agonist. ATR6.11 binding also enables complex formation between JSR1 and signaling partners. Our findings demonstrate the utility of retinal analogs for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)
. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 ...femtoseconds to the all-trans conformation
, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature
to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.
Abstract
While most plants die below a threshold of water content, desiccation-tolerant species display specific responses that allow them to survive extreme dehydration. Some of these responses are ...activated at critical stages during water loss and could represent the difference between desiccation tolerance (DT) and death. Here, we report the development of a simple and reproducible system to determine DT in Selaginella species. The system is based on exposure of excised tissue to a dehydration agent inside small containers, and subsequent evaluation for tissue viability. We evaluated several methodologies to determine viability upon desiccation including: triphenyltetrazolium chloride (TTC) staining, the quantum efficiency of PSII, antioxidant potential, and relative electrolyte leakage. Our results show that the TTC test is a simple and accurate assay to identify novel desiccation-tolerant Selaginella species, and can also indicate viability in other desiccation-tolerant models (i.e. ferns and mosses). The system we developed is particularly useful to identify critical points during the dehydration process. We found that a desiccation-sensitive Selaginella species shows a change in viability when dehydrated to 40% relative water content, indicating the onset of a critical condition at this water content. Comparative studies at critical stages could provide a better understanding of DT mechanisms and unravel insights into the key responses to survive desiccation.
A simple and efficient system to determine critical stages during dehydration in Selaginellathat can be applied to identify desiccation-tolerant plants.
Al nanohole array plasmonic biosensors have been fabricated on polycarbonate (PC) substrates from conventional compact discs (CD). Standard micro and nanofabrication processes have been used and ...optimized to be PC compatible. The viability of this CD-based plasmonic platform for label-free optical biosensing has been demonstrated through a competitive bioassay for biotin analysis using biotin-functionalized dextran-lipase conjugates immobilized on the transducer surface.
Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that make them highly suitable for (bio)sensing applications. Either as efficient light scattering centers ...for surface plasmon excitation or metal-clad optical waveguides, they are able to form strongly localized optical fields that can effectively interact with biomolecules and/or nanoparticles on the nanoscale. As the metal of choice, aluminum exhibits good optical and electrical properties, is easy to manufacture and process and, unlike gold and silver, its low cost makes it very promising for commercial applications. However, aluminum has been scarcely used for biosensing purposes due to corrosion and pitting issues. In this short review, we show our recent achievements on aluminum nanohole platforms for (bio)sensing. These include a method to circumvent aluminum degradation--which has been successfully applied to the demonstration of aluminum nanohole array (NHA) immunosensors based on both, glass and polycarbonate compact discs supports--the use of aluminum nanoholes operating as optical waveguides for synthesizing submicron-sized molecularly imprinted polymers by local photopolymerization, and a technique for fabricating transferable aluminum NHAs onto flexible pressure-sensitive adhesive tapes, which could facilitate the development of a wearable technology based on aluminum NHAs.
The in vitro activity of amikacin, gentamicin, kanamycin, tobramycin, neomycin, and netilmicin against 420 Escherichia coli producing extended spectrum β-lactamases (Ec-ESBLs) and 139 Klebsiella ...pneumoniae producing extended spectrum β-lactamase (Kp-ESBL) collected in two multicenter studies performed in Spain in 2000 and 2006 was determined. The presence of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methylases aac(3)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, ant(2")-Ia, ant(4')-IIa, aph(3')-Ia, aph(3')-IIa, armA, rmtB, and rmtC was also investigated. The resistance to (one or more) aminoglycosides was significantly higher in Kp-ESBL (104/139, 74.8%) than in Ec-ESBL (171/420, 40.7%; p < 0.0001). The lowest resistance rates for both species in the two studies were observed for amikacin. The prevalence of AME genes was significantly different in Ec-ESBL (161/420, 38.3%) than in Kp-ESBL (115/139, 82.7%; p < 0.0001). The most prevalent AME genes in Ec-ESBL and Kp-ESBL were aac(6')-Ib (16.2% and 44.6%) and aac(3)-IIa (14.7% and 43.1%), respectively. The expected phenotypic profile correlated with the found AMEs encoding genes in 59.6% Ec-ESBL and 26.1% Kp-ESBL. In Ec-ESBL, aac(6')-Ib was usually associated in 2000 with bla
(26.6%), but with bla
group (34.8%) in 2006, while aac(3)-IIa was coincident in 2000 with bla
(14.6%) and with bla
group (16.3%) in 2006. Among Kp-ESBL, the aac(6')-Ib and aac(3)-IIa genes were more frequent in strains with bla
(22.0% and 44.0%) in 2000 and with bla
group (46.4% and 34.0%) in 2006. Resistance to aminoglycosides in Ec-ESBL and Kp-ESBL is frequent and related to production of AMEs; this limits the clinical use of aminoglycosides against these organisms.
The electrochemical behavior of α-tetrathiophene (α-TTF) on Pt has been studied from concentrated monomer solutions in acetonitrile or mixtures of acetonitrile/nitrobenzene/DMF with 0.1 M LiClO
4. ...For a 1.0 mM α-TTF solution in 50:30:20 acetonitrile/nitrobenzene/DMF, three consecutive oxidation peaks are detected by cyclic voltammetry (CV). In this medium, uniform, insoluble, adherent and black polymeric films are obtained by chronopotentiometry (CP) until 0.5 mA cm
−2 and by chronoamperometry (CA) from 0.900 to 1.200 V versus Ag
∣
AgCl, corresponding to the first oxidation stage. Control voltammograms of films synthesized from 1.000 to 1.100 V show a redox pair related to electroactive polarons formed during poly(α-TTF) generation, followed by a reduction peak associated with bipolaronic states. Elemental analysis of polymers allows to determine that their monomeric units always support approximately 0.31 positive charges, balanced with ClO
4
− counterions, and contain a number of ionic couples of Li
+ClO
4
− ranging between 0.01 and
. 0.06. These results, along with the low productivity and high number of electrons required to incorporate one molecule of α-TTF into the polymer, points to the formation of crosslinked chains. This is consistent with the predominance of β–β linkages into the polymer, as confirmed by IR spectroscopy. Poly(α-TTF) doped with ClO
4
− ions has a very low conductivity, which is ascribed to the low number of polarons present in its monomeric units.
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