Essential autoproteolysis of bacterial anti-σ factor RsgI for transmembrane signal transduction.

Autoproteolysis has been discovered to play key roles in various biological processes, but functional autoproteolysis has been rarely reported for transmembrane signaling in prokaryotes. In this study, an autoproteolytic effect was discovered in the conserved periplasmic domain of anti-σ factor RsgIs from Clostridium thermocellum, which was found to transmit extracellular polysaccharide-sensing signals into cells for regulation of the cellulosome system, a polysaccharide-degrading multienzyme complex. Crystal and NMR structures of periplasmic domains from three RsgIs demonstrated that they are different from all known proteins that undergo autoproteolysis. The RsgI-based autocleavage site was located at a conserved Asn-Pro motif between the ß1 and ß2 strands in the periplasmic domain. This cleavage was demonstrated to be essential for subsequent regulated intramembrane proteolysis to activate the cognate SigI, in a manner similar to that of autoproteolysis-dependent activation of eukaryotic adhesion G protein-coupled receptors. These results indicate the presence of a unique prevalent type of autoproteolytic phenomenon in bacteria for signal transduction.

Integrated Sensing Chip for Ultrasensitive Label-Free Detection of the Products of Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that has been widely used for the detection of pathogens in many organisms. Current LAMP-based sensors usually require the LAMP products to be labeled in order for them to be detected. Here, we present a novel label-free LAMP chip, which consists of a nanopore thin-film sensor embedded inside a LAMP reaction chamber. A fraction of LAMP primers is immobilized on the sensor surface, allowing the LAMP products to be synthesized and bound to the sensor surface via immobilized primers. After the LAMP reaction components are removed from the reaction chamber, the amplified LAMP products bound to the sensor surface give rise to significantly increased transducing signals, which can be measured by a portable optical spectrometer through an optical fiber probe. As a demonstration, we used the LAMP chip to detect the causal agent of late blight, Phytophthora infestans, which is one of the most devastating plant pathogens and poses a major threat to sustainable crop production worldwide. We show that this chip can detect as low as 1 fg/µL of P. infestans DNA in 30 min, which corresponds to an attomolar level of 1.6 × 10-6 attomole/µL and is at least 10 times more sensitive than the currently available methods. This label-free sensing technology holds great promise to open up a new avenue for ultrasensitive, highly specific, rapid, and cost-effective point-of-care diagnostics of plant, animal, human, and foodborne pathogens.

Facile Process for Fabrication of Silicon Micro-Nanostructures of Different Shapes as Molds for Fabricating Flexible Micro-Nanostructures and Wearable Sensors.

We report a method to fabricate silicon micro-nanostructures of different shapes by tuning the number of layers and the sizes of self-assembled polystyrene beads, which serve as the mask, and by tuning the reactive ion etching (RIE) time. This process is simple, scalable, and inexpensive without using any sophisticated nanomanufacturing equipment. Specifically, in this work, we demonstrate the proposed process by fabricating silicon micro- or nanoflowers, micro- or nanobells, nanopyramids, and nanotriangles using a self-assembled monolayer or bilayer of polystyrene beads as the mask. We also fabricate flexible micro-nanostructures by using silicon molds with micro-nanostructures. Finally, we demonstrate the fabrication of bandage-type electrochemical sensors with micro-nanostructured working electrodes for detecting dopamine, a neurotransmitter related to stress and neurodegenerative diseases in artificial sweat. All these demonstrations indicate that the proposed process provides a low-cost, easy-to-use approach for fabricating silicon micro-nanostructures and flexible micro-nanostructures, thus paving a way for developing wearable micro-nanostructures enabled sensors for a variety of applications in an efficient manner.

Soft Contact Lens with Embedded Microtubes for Sustained and Self-Adaptive Drug Delivery for Glaucoma Treatment.

Because of the physiological and anatomical constraints of the eye, ophthalmic drug delivery is challenging. When applied topically, less than 1% of administered ophthalmic drugs reach the aqueous humor. The delivery of a drug within an efficient therapeutic concentration, to the required site of action, for an extended period of time, is complicated. Herein, a novel type of contact lens device, with embedded microtubes as drug containers, is reported. This device can provide a simple, noninvasive, extended drug release up to 45 days with higher bioavailability and lower risk for adverse effects. Another unique feature of the device is the release of drug triggered by stretching of the contact lens, indicating the possibility for achieving a self-adaptive drug release device for treating glaucoma patients.

Alternative σI/anti-σI factors represent a unique form of bacterial σ/anti-σ complex.

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of σI and anti-σI factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-σ factors that have N-terminal helical structures, RsgIN has a ß-barrel structure. Unlike other anti-σ factors that bind both σ2 and σ4 domains of the σ factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter -35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different σI/anti-σI pairs. We suggest that the σI/anti-σI factors represent a distinctive mode of σ/anti-σ complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate σI/anti-σI system.

High-resolution, flexible, and transparent nanopore thin film sensor enabled by cascaded Fabry-Perot effect.

This Letter reports a method to significantly improve the optical resolution of the anodic aluminum oxide (AAO) nanopore thin film sensor based on multi-cavity Fabry-Perot interference. The newly designed sensor is fabricated by bonding a layer of transparent polymer thin film (pTF), which is polydimethylsiloxane (PDMS), to a transparent AAO thin film to form a flexible pTF-nanopore sensor. In comparison with the AAO nanopore thin film sensor, the pTF-nanopore sensor shows a much-improved quality (Q) factor and optical resolution. Typical thicknesses of a PDMS layer and an AAO layer of the pTF-nanopore sensor are 80 µm and 2 µm, respectively. The pTF-nanopore sensor used for angle detection shows a sensitivity of 0.4 nm/deg with a resolution of 0.2 deg. The pTF-nanopore sensor can also be used for temperature monitoring with a sensitivity of 0.2 nm/°C and a resolution of 1°C.

Resonance assignments of the periplasmic domain of a cellulose-sensing trans-membrane anti-sigma factor from Clostridium thermocellum.

The cellulosome of Clostridium thermocellum is an elegant and efficient multi-enzyme complex for degrading lignocellulose. The cellulosome contains several dozens of carbohydrate hydrolysis enzymes, which are regulated by the presence of environmental substrates through several pairs of sigma and anti-sigma factors. The anti-sigma factors sense the presence of substrates and transduce the signals into the cell. The sigma factors are then released from the corresponding anti-sigma factors, and they recruit RNA polymerase to transcribe specific cellulosomal genes. However, it is not clear how the extracellular signals are transduced into the cell by the anti-sigma factors. The anti-sigma factors of C. thermocellum contain an N-terminal intracellular domain, a trans-membrane helix, a periplasmic domain, a proline-rich region which is probably required for crossing the cell wall, and a C-terminal carbohydrate-binding domain or glycoside hydrolase domain. The periplasmic domain may play a key role in signal transduction; however, its three-dimensional structure is still unknown. Here we report the NMR resonance assignments of the periplasmic domain of anti-sigma factor RsgI2 from C. thermocellum as a basis for further structural determination and functional studies.