Design and implementation of a low-cost multiparameter probe to evaluate the temporal variations of water quality conditions on an estuarine lagoon system.

The measurement of physicochemical variables to infer water quality is important since they help determine the distribution and abundance of aquatic organisms or pollution-related problems. Recently, the development of low-cost probes is a suitable alternative for continuous monitoring of these variables rather than the use of expensive instruments. In this work, a low-cost multiparameter probe (LCMP) has been developed to monitor water quality in an estuary located in Northwestern Mexico during a 3-month period. The LCMP integrates different sensors to an Arduino Nano microcontroller allowing to measure electrical conductivity, dissolved oxygen, pH, salinity, water temperature, and tide level. Data files were stored in a data logger system consisting of a secure digital (SD) card module and a real-time clock module coupled to the Arduino microcontroller. To ensure continuous operation, the system was powered by four 3.7 V, 10,000 mAh rechargeable LiPo batteries. All LCMP components were encapsulated in a polyvinyl chloride pipe. The results show that the LCMP had a good agreement with a commercial-grade multiparameter probe and was able to monitor continuously in hourly time steps. Finally, the LCMP proved to be an alternative for the establishment of coastal observatories, which has been deficient due to limited funding.

Effects of heat stress on pullet cloacal and body temperature.

One measure of the thermal status of poultry is cloacal temperature measured with a cloacal thermometer; however, this method requires handling the bird, is invasive, and can be stressful. Infrared thermography is an alternative means for assessing bird thermal status. The objective of this study was to investigate the body temperature response of pullets subjected to different environmental air temperatures during the growing phase and to evaluate the relationship between the cloacal temperature and the body parts surface temperature. A total of 648 chicks (Lohmann LSL Lite) were used in 2 different phases, phase I (day 1 through 6 wk of age) and phase II (week 7 through 17). During phase I, chicks were reared at 1 of 3 different thermal environments: thermal comfort (35°C-19°C), mild heat stress (38°C-22°C), or mild cold stress (28°C-17°C). In phase II, pullets were randomly redistributed to 1 of 4 daytime temperature treatments: 20°C; 25°C; 30°C; and 35°C, all with night time temperature of 20°C. Cloacal temperature and body surface temperature for 8 parts (head, eye, comb, chest, back, wing, leg, head area, and body area) were obtained weekly from 4 to 2 birds per treatment, respectively, during phase II. There were no effects for the interactions between the 2 experimental phases for cloacal and body parts surface temperature. There was a strong correlation (P < 0.001) between cloacal temperature and each body part temperature; cloacal temperature followed a quadratic response to environmental air temperature treatments. Pullets subjected to 35°C/20°C and 30°C/20°C had the highest body parts temperatures compared with the other 2 treatments (P < 0.05). The leg surface temperature was greatest in all treatments, and the chest the lowest. Regression between cloacal and body parts temperature had a 95% predictive accuracy of better than 0.4°C, suggesting a useful alternative to direct cloacal temperature measurement.

Productive performance and surface temperatures of Japanese quail exposed to different environment conditions at start of lay.

The objective of this study was to evaluate the effects of different environment conditions on productive performance and surface temperatures of Japanese quail (Coturnix coturnix japonica) during the initial stage of laying. In environmental controlled chambers, the birds were subjected to different temperatures and air velocities at the feeder. A total of 216 Japanese quails were distributed randomly in 2 galvanized wire cages, with 3 partitions each and 27 birds/cage. The experimental design consisted of randomized blocks with 2 treatments (air velocity at the feeder: 0, 1, 2, and 3 m/s and air temperature: 17, 23, 29, and 35°C) and 6 replicates. The productive performance was analyzed statistically (Sigma Plot 12.0) by 2-way ANOVA, with treatment means separated by the Tukey test (P < 0.05). To evaluate the main effects and interactions of the factors, the Holm-Sidak multiple comparisons test was performed using a mild condition as the control group (0 m/s). Feed intake did not differ (P > 0.05) among birds reared at temperatures of 23, 29, and 35°C, but higher feed intake was noted at 17°C. The mean values of egg production increased significantly (P < 0.05) with increased air velocity levels. It was observed that there was an increase in egg production and feed intake with the intensification of air velocity at the feeder, regardless of ambient temperature. Egg weight and feed conversion were not affected by air velocity treatments (P > 0.05). There was a significant positive correlation between air temperature and mean surface temperature and head surface temperature. In contrast, a significant negative correlation was observed between air velocity and mean surface temperature and head surface temperature. Productive performance was affected by temperature and air velocity, except for egg weight and feed conversion, which was not influenced by air velocity. Air velocity is important in removing heat from the surface of birds.

Effects of carbon dioxide on turkey poult performance and behavior.

Appropriate ventilation of poultry facilities is critical for achieving optimum performance. Ventilation promotes good air exchange to remove harmful gases, excessive heat, moisture, and particulate matter. In a turkey brooder barn, carbon dioxide (CO2) may be present at higher levels during the winter due to reduced ventilation rates to maintain high temperatures. This higher CO2 may negatively affect turkey poult performance. Therefore, the objective of this study was to evaluate the effects of subjecting tom turkey poults (commercial Large White Hybrid Converters) to different constant levels of atmospheric CO2 on their growth performance and behavior. In three consecutive replicate trials, a total of 552 poults were weighed post-hatch and randomly placed in 3 environmental control chambers, with 60 (Trial 1) and 62 (Trials 2 and 3) poults housed per chamber. They were reared with standard temperature and humidity levels for 3 wks. The poults were exposed to 3 different fixed CO2 concentrations of 2,000, 4,000, and 6,000 ppm throughout each trial. Following each trial (replicate), the CO2 treatments were switched and assigned to a different chamber in order to expose each treatment to each chamber. At the end of each trial, all poults were sent to a local turkey producer to finish growout. For each trial, individual body weight and group feed intake were measured, and mortality and behavioral movement were recorded. Wk 3 and cumulative body weight gain of poults housed at 2,000 ppm CO2 was greater (P < 0.05) than those exposed to 4,000 and 6,000 ppm CO2. Feed intake and feed conversion were unaffected by the different CO2 concentrations. No significant difference in poult mortality was found between treatments. In addition, no effect of CO2 treatments was evident in the incidence of spontaneous turkey cardiomyopathy for turkeys processed at 19 wk of age. Poults housed at the lower CO2 level (2,000 ppm) demonstrated reduced movement compared with those exposed to the 2 higher CO2 concentrations.

Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments.

By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles approximately 5-10 Kbp and traps approximately 0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.

Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies.

Atomic force microscopes and optical tweezers are widely used to probe the mechanical properties of individual molecules and molecular interactions, by exerting mechanical forces that induce transitions such as unfolding or dissociation. These transitions often occur under nonequilibrium conditions and are associated with hysteresis effects-features usually taken to preclude the extraction of equilibrium information from the experimental data. But fluctuation theorems allow us to relate the work along nonequilibrium trajectories to thermodynamic free-energy differences. They have been shown to be applicable to single-molecule force measurements and have already provided information on the folding free energy of a RNA hairpin. Here we show that the Crooks fluctuation theorem can be used to determine folding free energies for folding and unfolding processes occurring in weak as well as strong nonequilibrium regimes, thereby providing a test of its validity under such conditions. We use optical tweezers to measure repeatedly the mechanical work associated with the unfolding and refolding of a small RNA hairpin and an RNA three-helix junction. The resultant work distributions are then analysed according to the theorem and allow us to determine the difference in folding free energy between an RNA molecule and a mutant differing only by one base pair, and the thermodynamic stabilizing effect of magnesium ions on the RNA structure.

The effect of force on thermodynamics and kinetics: unfolding single RNA molecules.

We have used laser tweezers to unfold single RNA molecules at room temperature and in physiological-type solvents. The forces necessary to unfold the RNAs are over the range 10-20 pN, forces that can be generated by cellular enzymes. The Gibbs free energy for the unfolding of TAR (transactivation-responsive) RNA from HIV was found to be increased after the addition of argininamide; the TAR hairpin was stabilized. The rate of unfolding was decreased and the rate of folding was increased by argininamide.

Efficacy of directly observed treatment of HIV infection: experience in AIDS welfare homes.

With the objective of analyzing the efficacy of directly observed treatment (DOT) of HIV infection in the management of severely immunodepressed patients, this method was examined in individuals cared for in two social welfare facilities for HIV-infected persons and compared to self-administered therapy in outpatients. Forty-seven patients with registered HIV infection, stage C, were assigned to DOT for 9 months, the majority of whom had previously received antiretroviral therapy. A group of 51 HIV-infected outpatients, who attended day clinics attached to the reference hospitals, served as a comparison group. Together with increases in weight (9.2+/-7.5 kg) and Karnofsky scores (16.9+/-12.2) in the DOT group, a significant improvement of surrogate markers, such as CD4+ T-cell counts (increase in DOT group, 113.4+/-151.0 cells/microl; control group, -2.8+/-114.1 cells/microl; P<0.001) and HIV load (decrease in DOT group, -1.7+/-2.3 log10 copies/ml; control group, -0.4+/-1.5 log10 copies/ml; P<0.01) was detected in the DOT group. Morbidity and mortality were similar in both groups. The results indicate that such welfare facilities provide a useful framework not only for social objectives but also for healthcare purposes.

A two-state kinetic model for the unfolding of single molecules by mechanical force.

We investigate the work dissipated during the irreversible unfolding of single molecules by mechanical force, using the simplest model necessary to represent experimental data. The model consists of two levels (folded and unfolded states) separated by an intermediate barrier. We compute the probability distribution for the dissipated work and give analytical expressions for the average and variance of the distribution. To first order, the amount of dissipated work is directly proportional to the rate of application of force (the loading rate) and to the relaxation time of the molecule. The model yields estimates for parameters that characterize the unfolding kinetics under force in agreement with those obtained in recent experimental results. We obtain a general equation for the minimum number of repeated experiments needed to obtain an equilibrium free energy, to within k(B)T, from nonequilibrium experiments by using the Jarzynski formula. The number of irreversible experiments grows exponentially with the ratio of the average dissipated work, W(dis) to k(B)T.

Reversible unfolding of single RNA molecules by mechanical force.

Here we use mechanical force to induce the unfolding and refolding of single RNA molecules: a simple RNA hairpin, a molecule containing a three-helix junction, and the P5abc domain of the Tetrahymena thermophila ribozyme. All three molecules (P5abc only in the absence of Mg2+) can be mechanically unfolded at equilibrium, and when kept at constant force within a critical force range, are bi-stable and hop between folded and unfolded states. We determine the force-dependent equilibrium constants for folding/unfolding these single RNA molecules and the positions of their transition states along the reaction coordinate.

Formation of a GNRA tetraloop in P5abc can disrupt an interdomain interaction in the Tetrahymena group I ribozyme.

The secondary structure of a truncated P5abc subdomain (tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron ribozyme changes when its tertiary structure forms. We have now used heteronuclear NMR spectroscopy to determine its conformation in solution. The tP5abc RNA that contains only secondary structure is extended compared with the tertiary folded form; both forms coexist in slow chemical exchange (the interconversion rate constant is slower than 1 s(-1)) in the presence of magnesium. Kinetic experiments have shown that tertiary folding of the P5abc subdomain is one of the earliest folding transitions in the group I intron ribozyme, and that it leads to a metastable misfolded intermediate. Previous mutagenesis studies suggest that formation of the extended P5abc structure described here destabilize a misfolded intermediate. This study shows that the P5abc RNA subdomain containing a GNRA tetraloop in P5c (in contrast to the five-nucleotide loop P5c in the tertiary folded ribozyme) can disrupt the base-paired interdomain (P14) interaction between P5c and P2.

Structural and thermodynamic studies on mutant RNA motifs that impair the specificity between a viral replicase and its promoter.

The 3'-end region of the genomic RNA of brome mosaic virus forms a tRNA-like structure that is critical for its replication. Previous studies have shown that in this region, a stem-loop structure, called SLC, is necessary and sufficient for the binding of the RNA replicase, and for RNA replication. Recently, we determined the high-resolution NMR structure of SLC, which demonstrated that a 5'-AUA-3' triloop region is an important structural element for the enzymatic recognition. We proposed that the 5'-adenine of the triloop, which is rigidly fixed ("clamped") to the stem, is a key recognition element for the replicase. To elucidate the role of this "clamped base motif" for the enzymatic recognition, we have now investigated the solution conformations of several stem-loop molecules with mutant triloops, 5'-UUA-3', 5'-GUA-3', 5'-CUA-3' and 5'-UUU-3', that destroy the enzymatic recognition. For the GUA and UUA mutants, we have obtained high-resolution solution structures using 2D NMR. All four mutants have very similar thermodynamic stabilities, and all have the same secondary structures, a triloop with a five base-paired stem helix. In addition, they have quite similar sugar puckering patterns in the triloop region. The NMR structures of the GUA and UUA show that the 5' nucleotide of the triloop (G6 in GUA or U6 in UUA) lacks the strong interactions that hold its base in a fixed position. In particular, the U6 of UUA is found in two different conformations. Neither of these two mutants has the clamped base motif that was observed in the wild-type. While UUA also shows global change in the overall triloop conformation, GUA shows a very similar triloop conformation to the wild-type except for the lack of this motif. The absence of the clamped base motif is the only common structural difference between these two mutants and the wild-type. These results clearly indicate that the loss of function of the UUA and GUA mutants comes mainly from the destruction of a small key recognition motif rather than from global changes in their triloop conformations. Based on this study, we conclude that the key structural motif in the triloop recognized by the replicase is a solution-exposed, 5'-adenine base in the triloop that is clamped to the stem helix, which is called a clamped adenine motif.

Biophysical analysis of nucleic acids.

This overview unit provides a thorough overview of biophysical methods used for structure analysis, including X-ray diffraction, nuclear magnetic resonance, optical spectroscopy, theoretical and computational methods, and single-molecule methods. Advantages and disadvantages of the methods are compared.

Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA.

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.

A retroviral RNA kissing complex containing only two G.C base pairs.

The dimerization of viral RNA through noncovalent interactions at their 5' ends is a key step in the life cycle of retroviruses. In Moloney murine leukemia virus, three stem-loops are important in this process. One is a self-complementary tetraloop (H1), but the other two stem-loops (H2, H3) contain highly conserved GACG tetraloops that are not self-complementary sequences. Using two-dimensional NMR, we determined the structure of the H3 stem-loop. Surprisingly, it forms a stable, homodimeric kissing complex through only two intermolecular G small middle dotC base pairs. Cross-strand interactions of the adenines adjacent to the intermolecular G small middle dotC base pairs, plus unusual strong electrostatic interactions around the base pairs, contribute to the unexpected stability. This structure shows how even stem-loops without self-complementary sequences can facilitate the intermolecular recognition between two identical RNAs, and thus initiate dimerization and encapsidation of retroviral RNAs.

RNA motifs that determine specificity between a viral replicase and its promoter.

The 3' end of brome mosaic virus RNA contains a tRNA-like sequence that directs its RNA synthesis. A stem loop structure in this sequence, stem loop C (SLC), was investigated using NMR, and correlated with its ability to direct RNA synthesis by its replicase. SLC consists of two discrete domains, a flexible stem with an internal loop and a rigid stem containing a 5'-AUA-3' triloop. Efficient RNA synthesis requires the sequence on only one side of the flexible stem and a specific compact conformation of the triloop. A high resolution structure of the triloop places the 5' adenine out in solution, and the 3' adenine within the triloop, held tightly through stacking and unusual hydrogen bonds. This high resolution structure of an RNA promoter from a (+)-strand RNA virus provides new insights into how the RNA-dependent RNA polymerase binds to the RNA to initiate synthesis.

Solution structure of Cobalt(III)hexammine complexed to the GAAA tetraloop, and metal-ion binding to G.A mismatches.

The solution structure of a 22 nt RNA hairpin and its complex with Co(NH(3))(6)(3+) bound to the GAAA tetraloop has been determined by NMR spectroscopy. Co(NH(3))(6)(3+) has a similar geometry to Mg(H(2)O)(6)(2+) and can be used as a probe for binding sites of completely solvated magnesium ions. The hairpin contains tandem G.A mismatches, similar to the P5abc region of a group I intron, and is closed by a GAAA tetraloop. The tandem G.A mismatches are imino hydrogen bonded in contrast with the sheared G.A mismatches found in a different context in the crystal structure of the P4-P6 domain. Chemical shift changes of the imino protons upon titration of the RNA hairpin with Mg(2+) and with Co(NH(3))(6)(3+) were used to identify ion-binding sites. Paramagnetic resonance broadening upon titration with Mn(2+) was also used. The titration curves gave dissociation binding constants for the magnesium ions in the millimolar range, similar to the binding in the major groove of RNA at tandem G.U base-pairs. Although the largest chemical shift change occurred at an imino proton of one of the G.A base-pairs, no nuclear Overhauser enhancement cross-peaks between the cobalt ligand and neighboring RNA protons were seen, presumably due to the high mobility of the Co(NH(3))(6)(3+) at this site. Nuclear Overhauser enhancement cross-peaks between Co(NH(3))(6)(3+) and the GAAA tetraloop were observed, which allowed the determination of the structure of the tetraloop binding site. The Co(NH(3))(6)(3+) is bound in the major groove of the GAAA tetraloop with hydrogen bonds to guanine base N7 and to phosphate oxygen atoms of the tetraloop.

Assignment of cytosine N3 resonances in nucleic acids via intrabase three-bond coupling to amino protons.

Coherences were observed between 15N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with 15N. Use of intraresidue 15N3-amino proton couplings to assign cytosine 15N3 signals complements the recently proposed JNN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293-8297] method of identifying hydrogen-bonded base pairs in RNA.