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Rotational dynamics often challenge physical intuition while enabling unique realizations, from the rotor of a gyroscope that maintains its orientation regardless of the outer gimbals, to a tennis racket that rotates around its handle when tossed face-up in the air. In the context of inertial sensing, which can measure mass with atomic precision, rotational dynamics are normally considered a complication hindering measurement interpretation. Here, we exploit the rotational dynamics of a microfluidic device to develop a modality in inertial sensing. Combining theory with experiments, we show that this modality measures the volume of a rigid particle while normally being insensitive to its density. Paradoxically, particle density only emerges when fluid viscosity becomes dominant over inertia. We explain this paradox via a viscosity-driven, hydrodynamic coupling between the fluid and the particle that activates the rotational inertia of the particle, converting it into a 'viscous flywheel'. This modality now enables the simultaneous measurement of particle volume and mass in fluid, using a single, high-throughput measurement., (© 2021. The Author(s).)

Measuring the size of micron-scale particles plays a central role in the biological sciences and in a wide range of industrial processes. A variety of size parameters, such as particle diameter, volume, and mass, can be measured using electrical and optical techniques. Suspended microchannel resonators (SMRs) are microfluidic devices that directly measure particle mass by detecting a shift in resonance frequency as particles flow through a resonating microcantilever beam. While these devices offer high precision for sizing particles by mass, throughput is fundamentally limited by the small dimensions of the resonator and the limited bandwidth with which changes in resonance frequency can be tracked. Here, we introduce two complementary technical advancements that vastly increase the throughput of SMRs. First, we describe a deconvolution-based approach for extracting mass measurements from resonance frequency data, which allows an SMR to accurately measure a particle's mass approximately 16-fold faster than previously possible, increasing throughput from 120 particles/min to 2000 particles/min for our devices. Second, we describe the design and operation of new devices containing up to 16 SMRs connected fluidically in parallel and operated simultaneously on the same chip, increasing throughput to approximately 6800 particles/min without significantly degrading precision. Finally, we estimate that future systems designed to combine both of these techniques could increase throughput by nearly 200-fold compared to previously described SMR devices, with throughput potentially as high as 24 000 particles/min. We envision that increasing the throughput of SMRs will broaden the range of applications for which mass-based particle sizing can be employed.

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Microbes are an essential component of marine food webs and biogeochemical cycles, and therefore precise estimates of their biomass are of significant value. Here, we measured single-cell biomass distributions of isolates from several numerically abundant marine bacterial groups, including Pelagibacter (SAR11), Prochlorococcus and Vibrio using a microfluidic mass sensor known as a suspended microchannel resonator (SMR). We show that the SMR can provide biomass (dry mass) measurements for cells spanning more than two orders of magnitude and that these estimates are consistent with other independent measures. We find that Pelagibacterales strain HTCC1062 has a median biomass of 11.9±0.7 fg per cell, which is five- to twelve-fold smaller than the median Prochlorococcus cell's biomass (depending upon strain) and nearly 100-fold lower than that of rapidly growing V. splendidus strain 13B01. Knowing the biomass contributions from various taxonomic groups will provide more precise estimates of total marine biomass, aiding models of nutrient flux in the ocean.

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes.

Unlabelled: We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl3 to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability., Importance: Spores of Bacillus species cause food spoilage and disease and are extremely resistant to standard decontamination methods. This hardiness is partly due to spores' extremely low permeability to chemicals, including water. We present a method to directly monitor the uptake of molecules into B. subtilis spores by weighing spores in fluid. The results demonstrate the exchange of core water with subsecond resolution and show a correlation between water permeability and the rate at which small molecules can initiate or inhibit germination in coat-damaged spores. The ability to directly measure the uptake of molecules in the context of spores with known structural or genetic deficiencies is expected to provide insight into the determinants of spores' extreme resistance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.

The signaling capacity of seven-transmembrane/G-protein-coupled receptors (7TM/GPCRs) can be regulated through ligand-mediated receptor trafficking. Classically, the recycling of internalized receptors is associated with resensitization, whereas receptor degradation terminates signaling. We have shown previously that the incretin glucagon-like peptide-1 receptor (GLP-1R) internalizes fast and is primarily resensitized through recycling back to the cell surface. GLP-1R is expressed in pancreatic islets together with the closely related glucose-dependent insulinotropic polypeptide (GIPR) and glucagon (GCGR) receptors. The interaction and cross-talk between coexpressed receptors is a wide phenomenon of the 7TM/GPCR superfamily. Numerous reports show functional consequences for signaling and trafficking of the involved receptors. On the basis of the high structural similarity and tissue coexpression, we here investigated the potential cross-talk between GLP-1R and GIPR or GCGR in both trafficking and signaling pathways. Using a real-time time-resolved FRET-based internalization assay, we show that GLP-1R, GIPR, and GCGR internalize with differential properties. Remarkably, upon coexpression of the internalizing GLP-1R and the non-internalizing GIPR, GLP-1-mediated GLP-1R internalization was impaired in a GIPR concentration-dependent manner. As a functional consequence of such impaired internalization capability, GLP-1-mediated GLP-1R signaling was abrogated. A similar compromised signaling was found when GLP-1R internalization was abrogated by a dominant-negative version of dynamin (dynamin-1 K44E), which provides a mechanistic link between GLP-1R trafficking and signaling. This study highlights the importance of receptor internalization for full functionality of GLP-1R. Moreover, cross-talk between the two incretin receptors GLP-1R and GIPR is shown to alter receptor trafficking with functional consequences for GLP-1R signaling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell's buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell's water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell's dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density - the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein), we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell.

We present a general method to quantify coatings on microparticle surfaces based on the additional mass. Particle buoyant mass is determined in a solution with a density that is nearly equivalent to that of the core particle, reducing the magnitude and uncertainty of the measurement. Under these conditions, added material with a different density than that of the core is a larger fraction of the total buoyant mass of the coated particle. This method can resolve a buoyant mass difference between uncoated and coated particles of ~1 fg. For the protein layer on the 3 μm polystyrene spheres measured herein, this is equivalent to 1/10th of a full layer., (© 2012 American Chemical Society)

Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of cancer. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can directly quantify adsorbed mass via changes in resonant frequency. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from nonspecific binding. In this report, we demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR SiO(2) surfaces act as ultralow fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL.

We investigate the buoyant mass of bacterial cells in real time with the suspended microchannel resonator (SMR) as the population recovers from an osmotic shock. The density of the culture medium is chosen such that the bacteria initially have a positive buoyant mass which becomes negative as they recover from the hyperosmotic stress. This behavior can be used to differentiate between an antibiotic-resistant and an antibiotic-susceptible strain of the pathogenic bacteria Citrobacter rodentium, and we propose a general approach for exploiting the high precision of the SMR for rapid detection of antibiotic resistance.

This case report describes postoperative, reversible renal dysfunction and hypertension in a 5-year-old healthy boy after administration of relevant doses of ketorolac during anaesthesia. Two days postoperatively, the boy presented with polyuria, polydipsia, proteinuria, microscopic haematuria, and hypertension. He was treated with nifedipine. After six months the blood pressure and azotaemia parameters had normalised and antihypertensive treatment was discontinued.

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins and attract pharmaceutical interest as therapeutic targets. Two examples of class B GPCRs that are involved in metabolic diseases are the Parathyroid hormone receptor 1 (PTHR1) and the Glucagon-like-peptide-1 receptor (GLP-1R) which play central roles in osteoporosis and diabetes mellitus type II, respectively. Class B GPCRs are characterised by a large extracellular N-terminal domain with a typical disulfide bridge pattern. This domain is responsible for the binding of peptide hormone ligands. Here we report the recombinant expression of these ligands in natural and several modified forms for their use in functional assays, NMR analyses or affinity purification of receptor/ligand complexes for crystallisation. Applying the SUMO system, low cost expression of soluble fusion-proteins is achieved. Moreover, via the SUMO cleavage site, the authentic N-terminal sequence which is essential for ligand-receptor interactions can be obtained. Purification of the peptide by RP-HPLC results in >98% pure preparations. The strategy can also be adopted for many other purposes, especially if small peptides are needed at either large amounts or with specific features like isotope, affinity or fluorescent labels. Furthermore, for the growing demand for therapeutic peptides, this method could represent a straightforward production process.

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

Nanomechanical resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10(-21) g) have been weighed in vacuum, and proton-level resolution seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone-that is, with a high quality factor. In solution, viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnology and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resolution is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the solution inside a hollow resonator that is surrounded by vacuum. Here we demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resolution (1 Hz bandwidth). Central to these results is our observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of our silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resolution of six orders of magnitude over a high-end commercial quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the non-optical sizing and mass density measurement of colloidal particles.

Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. A variety of GLP-1 sequences are known from amphibian species, and some of these have been tested here and found to be able to bind and activate the human GLP-1 receptor. While little difference was observed for the in vitro potency for the human GLP-1 receptor, larger differences were found in the enzymatic stability of these peptides. Two peptides showed increased enzymatic stability, and they group together phylogenetically, though they originate from Amphibia and Reptilia. We have used ancestral sequence reconstruction to analyze the evolution of these GLP-1 molecules, including the synthesis of new peptides. We find that the increased stability could not be observed in the resurrected peptides from the common ancestor of frogs, even though they maintain the ability to activate the human GLP-1 receptor. Another method, using residue mapping on evolutionary branches yielded peptides that had maintained potency towards the receptor and also showed increased stability. This represents a new approach using evolutionary data in protein engineering.

DNAzymes are catalytically active DNA molecules, which have previously been described in solution. Here, we organize these molecules into a series of two-dimensional (2D) arrays using a periodic arrangement of DNA structures based on the DNA double crossover motif. We demonstrate by means of atomic force microscopy that the DNAzymes are organized according to the design and that they retain their activity when attached in linear strings within the context of the 2D array.

A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.

We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution.

With the aim of identifying structurally novel, centrally acting histamine H(3) antagonists, arrays of monoacyldiamines were screened. This led to the discovery of a series of 1-alkyl-4-acylpiperazines which were potent antagonists at the human histamine H(3) receptor. The most potent amides had antagonist potencies in the subnanomolar range.

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.

Background: Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially self-splicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo., Results: By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules., Conclusion: Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies.

The use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool.

Mammalian circadian rhythms generated in the hypothalamic suprachiasmatic nuclei are entrained to the environmental light/dark cycle via a monosynaptic pathway, the retinohypothalamic tract (RHT). We have shown previously that retinal ganglion cells containing pituitary adenylate cyclase-activating polypeptide (PACAP) constitute the RHT. Light activates the RHT via unknown photoreceptors different from the classical photoreceptors located in the outer retina. Two types of photopigments, melanopsin and the cryptochromes (CRY1 and CRY2), both of which are located in the inner retina, have been suggested as "circadian photopigments." In the present study, we cloned rat melanopsin photopigment cDNA and produced a specific melanopsin antibody. Using in situ hybridization histochemistry combined with immunohistochemistry, we demonstrate that the distribution of melanopsin was identical to that of the PACAP-containing retinal ganglion cells. Colocalization studies using the specific melanopsin antibody and/or cRNA probes in combination with PACAP immunostaining revealed that melanopsin was found exclusively in the PACAP-containing retinal ganglion cells located at the surface of somata and dendrites. These data, in conjunction with published action spectra analyses and work in retinally degenerated (rd/rd/cl) mutant mice, suggest that melanopsin is a circadian photopigment located in retinal ganglion cells projecting to the biological clock.

The conserved residues Y239 and L240 of human VPAC1 receptor are predicted to be at the same location as the asparagine and arginine in the "DRY" motif in the Rhodopsin family of G protein-coupled receptors. By comparing vasoactive intestinal peptide (VIP) binding with or without the presence of GTP-gamma-S, it was found that the deltadelta G(o) for the endogenous G-protein coupling was 1.5 kJ/mol, 0.95 kJ/mol, and 3.4 kJ/mol for theY239A, L240A, and wild-type receptor, respectively. VIP-induced cAMP production in whole cells support the results of the binding studies, as Y239A had a moderate and L240A a pronounced impaired ability to produce cAMP. The mutants had a minor influence on the intrinsic "low affinity to high affinity equilibrium," suggesting that the dominating effect of these mutants is a perturbation of the G protein-binding site. Thus, the highly diverged chemical properties of the hydrophobic "YL" motif and charged "DR(Y)" motif could be a crucial difference between the Secretin Receptor Family and the Rhodopsin Family with respect to receptor activation and G-protein coupling.

The importance of three conserved transmembrane prolines of the human vasoactive intestinal polypeptide (VPAC)(1) receptor was examined by single alanine substitution. P266A, P300A and P348A reduced the expression level, but maintained the binding to VIP. P266A showed decreased ability to stimulate cAMP, while P300A and P348A displayed an increased potency in cAMP production combined with a high sensitivity towards GTP compared to the wild type receptor. In addition, substitutions of two conserved leucines located in position -2 and +1 from P348 were investigated. L346A and L349A reduced the receptor expression, influenced the G protein coupling and decreased the receptor activity. These observations, which are the first on conserved transmembrane prolines within this family of receptors, indicate that these residues are important for receptor expression, G protein coupling and receptor activity.

The suprachiasmatic nucleus generates circadian rhythms which are synchronized to the environmental light-dark cycle via the retinohypothalamic tract. Pituitary adenylate cyclase-activating polypeptide and glutamate, two neurotransmitters co-stored in the retinohypothalamic tract of the rat, are able to phase shift the endogenous rhythm similar to light. The "clock genes" period1 (per1) and per2, which show circadian oscillation within the suprachiasmatic nucleus, have been attributed a role in light-induced resetting of the mammalian circadian clock due to rapid induction of the period (per) genes after light stimulation at night. Using a rat in vitro brain slice model, we demonstrate by quantitative in situ hybridization histochemistry that the diurnal alteration in expression of both per genes in the suprachiasmatic nucleus was retained in vitro. In the model, we examined the effects of pituitary adenylate cyclase-activating polypeptide and glutamate alone and in combination on per1 and per2 gene expression at late subjective night (circadian time 19). Glutamate administration (10(-3)M) induced both per1 and per2 gene expression in the suprachiasmatic nucleus of the brain slice within 1h. The per gene responses were similar to the induction of gene expression observed after light stimulation in vivo at late night. Pituitary adenylate cyclase-activating polypeptide (10(-6)M) administered alone had no effect on the per gene expression, but when pituitary adenylate cyclase-activating polypeptide in micromolar concentration was applied before glutamate, the neuropeptide blocked the glutamate-induced per1 and per2 gene expression in the suprachiasmatic nucleus. In contrast to the lack of effect of pituitary adenylate cyclase-activating polypeptide itself in micromolar concentration, pituitary adenylate cyclase-activating polypeptide (10(-9)M) induced both per1 and per2 gene expression, an effect which was not augmented by co-application of glutamate. Our results provide the molecular substrate for the previous electrophysiological findings that pituitary adenylate cyclase-activating polypeptide in high concentration is able to block glutamate-induced phase advance at late night, and that the peptide in low concentration can induce a phase advance similar to light and glutamate.

Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HDV contain a ribozyme that undergoes cis-cleavage, thereby processing the transcript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have attenuated self-cleavage. Structure mapping and site-directed mutagenesis revealed an inhibitory stretch consisting of upstream nucleotides -24 to -15 that forms a long-range pairing, termed Alt 1, with the 3' strand of P2 (P2(3')) located at the very 3'-end of the ribozyme. Two other alternative pairings were found, Alt 2, which involves upstream nucleotide-ribozyme interactions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleavage was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the -24/-15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased to 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides -54 to -18, termed P(-1)/L(-1), which sequesters the majority of the -24/-15 inhibitory stretch in cis. This hairpin is followed by a stretch of single-stranded pyrimidine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, and that the J(-1/1) stretch is conserved among antigenomic HDV isolates. Lastly, the secondary structure of the Alt 1-containing ribozyme provides insight into possible folding intermediates of the ribozyme., (Copyright 2000 Academic Press.)

To elucidate the functional role of the second extracellular loop of human vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide (VIP/PACAP) receptor (hVPAC1R), surface expression, ligand binding, and receptor activation were analyzed. Amino acids in the entire second extracellular loop were individually substituted by alanine by site-directed mutagenesis. The mutant and wild-type receptors were transiently expressed in HEK293 cells and purified cell membranes were tested for the ability to bind VIP, while the receptor activity was measured as potency of cAMP production analysed on intact cells. Surface expression of the substituted conserved residues, W286A, I289A, W294A, and W295A, was evidently decreased to 20-30% compared to the wild-type expression. W286A also showed an significantly reduced potency of cAMP production. Substituted residues as F280A, E281A, and G284A showed a significant reduction in the potency of stimulated cAMP production amounting to 8-46-fold, compared to the wild-type with unaffected surface expression and VIP binding. These results indicate that some residues in the second extracellular loop of the human VPAC1R participate in the active mechanism of a ligand-mediated response without being directly involved in the binding of VIP.

The importance of two highly conserved cysteines in the human pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide 1 (VIP1) receptor was examined. Using site-directed mutagenesis, each Cys residue was converted into Ala or Ser. The mutant and wildtype genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215Ala/Ser and Cys285Ala/Ser showed at least a tenfold decrease in binding affinity and receptor potency when compared to the wildtype. In contradiction to the wildtype receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilizing the receptor in the correct conformation for ligand binding and activation.

An arrangement of the seven transmembrane alpha-helices of the G protein-coupled Secretin receptor family is proposed. The helices of 27 homologous receptor sequences were plotted as helical wheels. The solvent inaccessible portion of each helix was used to assign relative orientations. They were arranged according to two criteria: 1) conserved, hydrophilic residues and aligned positions with restricted volume changes face the other helices and 2) aligned positions with low identity and large volume change face the lipid. The positive inside rule confirms the assumption that loops connecting transmembrane helices I-II, III-IV, V-VI and the C-terminal part of the receptors are intracellular. Our model approach was tested using the Bacteriorhodopsin family. The use of volume changes at each position in the transmembrane helix was crucial for the good correlation of the orientation of the helices using the model approach and the structure of bacteriorhodopsin solved by electron microscopy [Grigorieff N, Ceska TA, Downing KH, Baldwin JM, and Henderson R (1996) J Mol Biol 259 393-421]. The tests of our modelling approach showed that six helices were within a 15 degrees derivation in the orientation and five helices were within a horizontal derivation of two residues. The largest orientational derivations of a helix were 40 degrees and the largest horizontal displacement was four residues. A long stretch of side chains predicted to possess low resistance to movement in helix V of the Secretin receptor family suggests an involvement in receptor activation. Comparison of the Secretin receptor family and the larger G protein-coupled Rhodopsin family showed many similarities, despite the lack of obvious sequence identity.

The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor I (hVIPR 1) was examined. By site-directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild-type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215-Ala/Ser and Cys285-Ala/Ser showed at least a 10-fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild-type receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilising the receptor in the correct conformation for ligand binding and activation.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the VIP (vasoactive intestinal polypeptide) family of peptides, has been demonstrated in neurons of the sensory system. PACAP expression of these neurons is sensitive to nerve damages such as nerve crush or axotomy. In the present study, PACAP expression in the mesencephalic trigeminal nucleus of the rat was examined after transsection of the main trunk of the masseteric nerve. The primary sensory neurons of the nucleus are considered to have purely proprioceptive functions. By quantitative in situ hybridization using a PACAP [35S]cRNA probe, an increase in PACAP mRNA was observed on the side ipsilateral to transsection already after 3 h and the expression reached a peak 24 h after surgery after which the levels gradually decreased during the next 14 days. A low and constant expression of PACAP mRNA could be seen on the side contralateral to transsection. PACAP immunoreactivity was demonstrated on the ipsilateral side after 18 h, using a specific monoclonal PACAP antibody. Co-existence of PACAP with NPY and galanin was demonstrated 7 days after transsection. Analysis of the masseteric nerve by radioimmunoassay on transsected and normal nerve stumps revealed an increase of PACAP-38 immunoreactivity in the nerve proximal to the transsection compared to the normal side (15.3 vs. 6.1 pmol/g wt). The results suggest that PACAP has a role in the early phase of adaptation to nerve injury in the proprioceptive neurons.

The human elongation factor 1alpha gene family consists of at least 2 actively transcribed genes, EEF1A and EEF1A2, and more than 18 homologous loci. EEF1A2 is expressed in a tissue-specific manner, whereas EEF1A is expressed ubiquitously, and both of them can function in translation. An EEF1A cDNA probe has previously been shown to cross-hybridize with several human chromosomes, but the location of the functional gene has not been established. We have mapped the functional EEF1A gene to 6q14 by combined fluorescence in situ hybridization (FISH) and PCR analysis of a somatic cell hybrid panel and mapped EEF1A2 to 20q13.3 by FISH. In addition, the 11 strongest cross-hybridizing loci (EEF1AL2-EEF1AL13) were mapped by FISH to 12p12, 9q34, 7p15-p21, 19q13, 3q26-q27, 7q33-q35, 1p13-p22, 2q12-q14, 5p12-q11, 1q31-q32, and Xq21.

A novel isoform of human elongation factor-1 alpha (EF-1 alpha 2) has been characterised. It shows a high similarity to other EF-1 alpha proteins, especially to a rat EF-1 alpha variant and it has all the characteristics of a functional EF-1 alpha protein. The pattern of expression of both EF-1 alpha 2 and EF-1 alpha was analysed in different human tissues. This showed that the two proteins were differentially expressed, EF-1 alpha 2 was expressed in brain, heart, skeletal muscle and in the transformed cell lines AMA and K14, but was undetectable in other tissues and in both primary and transformed human fibroblasts. EF-1 alpha was expressed in brain, placenta, lung, liver, kidney, pancreas and in all the cell lines that we have analysed but barely detectable in heart and skeletal muscle.

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.