Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying.

2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

A Facile and General Tandem Oligonucleotide Synthesis Methodology for DNA and RNA.

Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of an immolative linker within a single sequence composed of multiple oligonucleotide fragments. Here, we report the use of a commercially available building block, typically utilized for the chemical phosphorylation of DNA/RNA oligomers, to perform TOS. We show that the 2,2'-sulfonyldiethylene linker is efficiently self-immolated during the standard deprotection of DNA and RNA and presents itself as a generalizable methodology for nucleic acid TOS. Furthermore, we show the utility of this methodology by assembling a model siRNA construct, and showcase a template-directed ligation pathway to incorporate phosphoramidate or pyrophosphate linkages within DNA oligomers.

Ultrasound-Guided Dorsolateral Approach for Quadratus Lumborum Block in Rabbits (Oryctolagus cuniculus): A Prospective, Randomized, Blinded, Cadaveric Study Comparing Four Different Injectate Volumes.

Ultrasound-guided (US-guided) loco-regional anesthesia can provide significant analgesia and anesthetic-sparing effects when used in rabbits. The aims of this study were to investigate the thoraco-lumbar anatomy of the rabbits, particularly the quadratus lumborum (QL) muscle, to design an appropriate US-guided quadratus lumborum block (QLB) specific for rabbits, and to define the most adequate volume of injectate required to consistently cover the ventral branches of T11 to L3 without affecting the pelvic limb innervation (L4, L5 and L6). Sixteen adult rabbit cadavers were included in the study. After randomization, four different volumes of injectate (0.1 mL/kg, 0.2 mL/kg, 0.3 mL/kg and 0.4 mL/kg) were tested, with these volumes additionally randomized to two sites of injection (right or left QL fascia). An ultrasound-guided QLB was performed with a solution of lidocaine, iodinated contrast and tissue dye (in a proportion of 3:1:1 volume, respectively), with subsequent computed tomography (CT) and anatomical dissection, to evaluate the spread of the injectate. In all but one case, the US-guided QLB performed with a dorsolateral approach using 0.3 mL/kg was adequate, while a dose of 0.4 mL/kg consistently reached the targeted nerves but also extended to L4 and caudally. This may suggest that an injectate volume of 0.3 mL/kg may be the most appropriate to produce adequate spread while not affecting pelvic limb innervation.

Comparison of the effects of propofol and alfaxalone on the electrocardiogram of dogs, with particular reference to QT interval.

Cardiac electrical activity is often altered by administration of anesthetic drugs. While the effects of propofol in this regard have previously been described in dogs, to date, there are no reports of the effect of alfaxalone. This study investigated the impact of both propofol and alfaxalone on the ECG of 60 dogs, after premedication with acepromazine and methadone. Heart rate increased significantly in both groups. The PR and QRS intervals were significantly increased following propofol while with alfaxalone the QRS duration was significantly increased and ST segment depression was observed. The QT and JT interval were significantly shorter following induction with alfaxalone, but, when corrected (c) for heart rate, QTc and JTc in both groups were significantly greater following induction. When comparing the magnitude of change between groups, the change in RR interval was greater in the alfaxalone group. The change in both QT and JT intervals were significantly greater following alfaxalone, but when QTc and JTc intervals were compared, there were no significant differences between the two drugs. The similarly increased QTc produced by both drugs may suggest comparable proarrhythmic effects.

Correction: Felisberto et al. Ultrasound-Guided Saphenous Nerve Block in Rabbits (Oryctolagus cuniculus): A Cadaveric Study Comparing Two Injectate Volumes. Animals 2022, 12, 624.

The authors wish to make the following correction to this paper [...].

Ultrasound-Guided Saphenous Nerve Block in Rabbits (Oryctolagus cuniculus): A Cadaveric Study Comparing Two Injectate Volumes.

Ultrasound-guided (US-guided) loco-regional anesthesia techniques allow direct visualization and blockade of sensory nerves. The saphenous nerve (SN), a terminal branch of the femoral nerve (FN), is strictly a sensory nerve for which electrical locator devices are ineffective for localization as no effector muscle contractions can be evoked. US-guided SN block in species other than rabbits produces hind-limb analgesia without affecting FN motor function. The aims of this study were to develop a US-guided SN block technique in rabbits and to compare the spread obtained using two different dye volumes. Twelve hind-limbs from six cadavers (1.62 ± 0.1 kg) were included; after randomization, the SN block was performed on the right or left hind-limb, injecting 0.05 mL kg-1 or 0.1 mL kg-1 of tissue dye in lidocaine (1:50 v:v). Subsequent dissections allowed nerve staining measurements. All SNs were identified, and 17.8 ± 4.6% and 31.0 ± 8.9% of the SN length were stained using low-volume and high-volume of the dye, respectively. Regardless of the volume used, the SN was consistently stained while the motor branch of the FN was not. This US-guided technique may provide hind-limb analgesia without affecting FN motor function in rabbits undergoing mid-distal hind-limb surgeries.

Validation of oscillometric blood pressure measurement using a Datex S/5 Compact multiparameter monitor in anaesthetized adult dogs.

OBJECTIVE: To compare noninvasive (NIBP) with invasive blood pressure (IBP) measurements from a Datex S/5 Compact monitor in anaesthetized adult dogs, and to evaluate it according to the American College of Veterinary Internal Medicine (ACVIM) and the Association for the Advancement of Medical Instrumentation (AAMI) criteria. STUDY DESIGN: Prospective clinical study. ANIMALS: A group of 34 client-owned adult dogs. METHODS: Dogs were anaesthetized for different surgical procedures using different anaesthetic protocols. IBP was measured using a catheter placed in a dorsal pedal artery. A blood pressure cuff was placed over the contralateral dorsal pedal artery for NIBP measurement. Data were recorded using the Datex iCollect program, and paired readings were matched every 3 minutes for 60 minutes. Bland-Altman and error grid analyses were used to estimate the agreement between IBP and NIBP measurements, and its clinical significance, respectively. Data were reported as mean bias [lower, upper limits of agreement (LoA)]. RESULTS: The Datex S/5 monitor conformed to most ACVIM criteria. The correlation coefficient was less than 0.9 for systolic, diastolic, and mean arterial pressures (MAP). The best agreement between the noninvasive and invasive methods was observed for MAP, with LoA (-17 to 13 mmHg) and higher percentage of NIBP readings within 5 (55.6%), 10 (81.7%) and 20 (98.6%) mmHg of the IBP values. The Datex S/5 NIBP technology did not meet the AAMI validation criteria and less than 95% of the paired measurements were found within the green zone of the error grid analysis. CONCLUSIONS AND CLINICAL RELEVANCE: The Datex S/5 monitor conformed to most ACVIM criteria but not with the more rigorous AAMI standards. Despite good agreement between IBP and NIBP for MAP measurements, care must be taken when using this device to guide therapeutic interventions of blood pressure in anaesthetized healthy adult dogs.

Accuracy and trending capability of haemoglobin measurement by noninvasive pulse co-oximetry in anaesthetized horses.

OBJECTIVE: To assess the accuracy and trending capability of continuous measurement of haemoglobin concentration [Hb], haemoglobin oxygen saturation (SaO2) and oxygen content (CaO2) measured by the Masimo Radical-7 pulse co-oximeter in horses undergoing inhalational anaesthesia. STUDY DESIGN: Prospective observational clinical study. ANIMALS: A group of 23 anaesthetized adult horses. METHODS: In 23 healthy adult horses undergoing elective surgical procedures, paired measurements of pulse co-oximetry-based haemoglobin concentration (SpHb), SaO2 (SpO2), and CaO2 (SpOC) and simultaneous arterial blood samples were collected at multiple time points throughout anaesthesia. The arterial samples were analysed by a laboratory co-oximeter for total haemoglobin (tHb), SaO2 and manually calculated CaO2. Bland-Altman plots, linear regression analysis, error grid analysis, four-quadrant plot and Critchley polar plot were used to assess the accuracy and trending capability of the pulse co-oximeter. Data are presented as mean differences and 95% limits of agreement (LoA). RESULTS: In 101 data pairs analysed, the pulse co-oximeter slightly underestimated tHb (bias 0.06 g dL-1; LoA -1.0 to 1.2 g dL-1), SaO2 (bias 1.4%; LoA -2.0% to 4.8%), and CaO2 (bias 0.3 mL dL-1; LoA -2.1 to 2.7 mL dL-1). Zone A of the error grid encompassed 99% of data pairs for SpHb. Perfusion index (PI) ≥ 1% was recorded in 58/101 and PI < 1% in 43/101. The concordance rate for consecutive changes in SpHb and tHb with PI ≥ 1% and < 1% was 80% and 91% with four-quadrant plot, and 45.8% and 66.6% with Critchley polar plot. CONCLUSIONS: Pulse co-oximetry has acceptable accuracy for the values measured, even with low PI, whereas its trending ability requires further investigation in those horses with a higher [Hb] variation during anaesthesia.

Thermally Driven Membrane Phase Transitions Enable Content Reshuffling in Primitive Cells.

Self-assembling single-chain amphiphiles available in the prebiotic environment likely played a fundamental role in the advent of primitive cell cycles. However, the instability of prebiotic fatty acid-based membranes to temperature and pH seems to suggest that primitive cells could only host prebiotically relevant processes in a narrow range of nonfluctuating environmental conditions. Here we propose that membrane phase transitions, driven by environmental fluctuations, enabled the generation of daughter protocells with reshuffled content. A reversible membrane-to-oil phase transition accounts for the dissolution of fatty acid-based vesicles at high temperatures and the concomitant release of protocellular content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled material in a new cohort of protocells. Notably, we find that our disassembly/reassembly cycle drives the emergence of functional RNA-containing primitive cells from parent nonfunctional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally driven tunable prebiotic process, which supports the release and reshuffling of oligonucleotides and membrane components, potentially leading to a new generation of protocells with superior traits. In the absence of protocellular transport machinery, the environmentally driven disassembly/assembly cycle proposed herein would have plausibly supported protocellular content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early life forms.

Ribose Alters the Photochemical Properties of the Nucleobase in Thionated Nucleosides.

Substitution of exocyclic oxygen with sulfur was shown to substantially influence the properties of RNA/DNA bases, which are crucial for prebiotic chemistry and photodynamic therapies. Upon UV irradiation, thionucleobases were shown to efficiently populate triplet excited states and can be involved in characteristic photochemistry or generation of singlet oxygen. Here, we show that the photochemistry of a thionucleobase can be considerably modified in a nucleoside, that is, by the presence of ribose. Our transient absorption spectroscopy experiments demonstrate that thiocytosine exhibits 5 times longer excited-state lifetime and different excited-state absorption features than thiocytidine. On the basis of accurate quantum chemical simulations, we assign these differences to the dominant population of a shorter-lived triplet nπ* state in the nucleoside and longer-lived triplet ππ* states in the nucleobase. This explains the distinctive photoanomerziation of thiocytidine and indicates that the nucleoside will be a less efficient phototherapeutic agent with regard to singlet oxygen generation.

Comparison between dexmedetomidine and acepromazine in combination with methadone for premedication in brachycephalic dogs undergoing surgery for brachycephalic obstructive airway syndrome.

OBJECTIVE: To compare dexmedetomidine with acepromazine for premedication combined with methadone in dogs undergoing brachycephalic obstructive airway syndrome (BOAS) surgery. STUDY DESIGN: Randomized, blinded clinical study. ANIMALS: A group of 40 dogs weighing mean (± standard deviation) 10.5 ± 6 kg, aged 2.6 ± 1.9 years. METHODS: Dogs received either acepromazine 20 µg kg-1 (group A) or dexmedetomidine 2 µg kg-1 (group D) intramuscularly with methadone 0.3 mg kg-1. Anaesthesia was induced with propofol and maintained with sevoflurane. Sedation (0-18), induction (0-6) and recovery (0-5) qualities were scored. Propofol dose, hypotension incidence, mechanical ventilation requirement, extubation time, additional sedation, oxygen supplementation, regurgitation and emergency intubation following premedication or during recovery were recorded. Data were analysed using t tests, Mann-Whitney U or Chi-square tests. RESULTS: Group A dogs were less sedated [median (range): 1.5 (0-12)] than group D [5 (1-18)] (p = 0.021) and required more propofol [3.5 (1-7) versus 2.4 (1-8) mg kg-1; p = 0.018]. Induction scores [group A: 5 (4-5); group D 5 (3-5)] (p = 0.989), recovery scores [group A 5 (4-5); group D 5(3-5)](p = 0.738) and anaesthesia duration [group A:93 (50-170); group D 96 (54-263) minutes] (p = 0.758) were similar between groups. Time to extubation was longer in group A 12.5 (3-35) versus group D 5.5 (0-15) minutes; (p = 0.005). During recovery, two dogs required emergency intubation (p > 0.99) and five dogs required additional sedation (p > 0.99). Oxygen supplementation was required in 16 and 12 dogs in group A and D, respectively (p = 0.167); no dogs in group A and one dog in group D regurgitated (p = 0.311). CONCLUSIONS AND CLINICAL RELEVANCE: Dexmedetomidine 2 µg kg-1 produces more sedation but similar recovery quality to acepromazine 20 µg kg-1 combined with methadone in dogs undergoing BOAS surgery.

Assessment of pulse co-oximetry technology after in vivo adjustment in anaesthetized dogs.

OBJECTIVE: To compare values of haemoglobin concentration (SpHb), arterial haemoglobin saturation (SpO2) and calculated arterial oxygen content (SpOC), measured noninvasively with a pulse co-oximeter before and after in vivo adjustment (via calibration of the device using a measured haemoglobin concentration) with those measured invasively using a spectrophotometric-based blood gas analyser in anaesthetized dogs. STUDY DESIGN: Prospective observational clinical study. ANIMALS: A group of 39 adult dogs. METHODS: In all dogs after standard instrumentation, the dorsal metatarsal artery was catheterised for blood sampling, and a pulse co-oximeter probe was applied to the tongue for noninvasive measurements. Paired data for SpHb, SpO2 and SpOC from the pulse co-oximeter and haemoglobin arterial oxygen saturation (SaO2) and arterial oxygen content (CaO2) from the blood gas analyser were obtained before and after in vivo adjustment. Bland-Altman analysis for repeated measurements was used to evaluate the bias, precision and agreement between the pulse co-oximeter and the blood gas analyser. Data are presented as mean differences and 95% limits of agreement (LoA). RESULTS: A total of 39 data pairs were obtained before in vivo adjustment. The mean invasively measured haemoglobin-SpHb difference was -2.7 g dL-1 with LoA of -4.9 to -0.5 g dL-1. After in vivo adjustment, 104 data pairs were obtained. The mean invasively measured haemoglobin-SpHb difference was -0.2 g dL-1 with LoA of -1.1 to 0.6 g dL-1. The mean SaO2-SpO2 difference was 0.86% with LoA of -0.8% to 2.5% and that between CaO2-SpOC was 0.66 mL dL-1 with LoA of -2.59 to 3.91 mL dL-1. CONCLUSIONS: Before in vivo adjustment, pulse co-oximeter derived values overestimated the spectrophotometric-based blood gas analyser haemoglobin and CaO2 values. After in vivo adjustment, the accuracy, precision and LoA markedly improved. Therefore, in vivo adjustment is recommended when using this device to monitor SpHb in anaesthetised dogs.

The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis.

Recent advances in prebiotic chemistry are beginning to outline plausible pathways for the synthesis of the canonical ribonucleotides and their assembly into oligoribonucleotides. However, these reaction pathways suggest that many noncanonical nucleotides are likely to have been generated alongside the standard ribonucleotides. Thus, the oligomerization of prebiotically synthesized nucleotides is likely to have led to a highly heterogeneous collection of oligonucleotides comprised of a wide range of types of nucleotides connected by a variety of backbone linkages. How then did relatively homogeneous RNA emerge from this primordial heterogeneity? Here we focus on nonenzymatic template-directed primer extension as a process that would have strongly enriched for homogeneous RNA over the course of multiple cycles of replication. We review the effects on copying the kinetics of nucleotides with altered nucleobase and sugar moieties, when they are present as activated monomers and when they are incorporated into primer and template oligonucleotides. We also discuss three variations in backbone connectivity, all of which are nonheritable and regenerate native RNA upon being copied. The kinetic superiority of RNA synthesis suggests that nonenzymatic copying served as a chemical selection mechanism that allowed relatively homogeneous RNA to emerge from a complex mixture of prebiotically synthesized nucleotides and oligonucleotides.

siRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

Multicenter study to investigate factors associated with change in rectal temperature during anesthesia in dogs.

OBJECTIVE: To determine factors associated with change in rectal temperature (RT) of dogs undergoing anesthesia. ANIMALS: 507 dogs. PROCEDURES: In a prospective observational study, the RT of dogs undergoing anesthesia at 5 veterinary hospitals was recorded at the time of induction of anesthesia and at the time of recovery from anesthesia (ie, at the time of extubation). Demographic data, body condition score, American Society of Anesthesiologists (ASA) physical status classification, types of procedure performed and medications administered, duration of anesthesia, and use of heat support were also recorded. Multiple regression analysis was performed to determine factors that were significantly associated with a decrease or an increase (or no change) in RT. Odds ratios were calculated for factors significantly associated with a decrease in RT. RESULTS: Among the 507 dogs undergoing anesthesia, RT decreased in 89% (median decrease, -1.2°C [-2.2°F]; range, -0.1°C to -5.7°C [-0.2°F to -10.3°F]), increased in 9% (median increase, 0.65°C [1.2°F]; range, 0.1°C to 2.1°C [3.8°F]), and did not change in 2%. Factors that significantly predicted and increased the odds of a decrease in RT included lower weight, ASA classification > 2, surgery for orthopedic or neurologic disease, MRI procedures, use of an α2-adrenergic or µ-opioid receptor agonist, longer duration of anesthesia, and higher heat loss rate. Lack of µ-opioid receptor agonist use, shorter duration of anesthesia, and lower heat loss rate were significantly associated with an increase in RT. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple factors that were associated with a decrease in RT in dogs undergoing anesthesia were identified. Knowledge of these factors may help identify dogs at greater risk of developing inadvertent perianesthetic hypothermia.

Assembly of a Ribozyme Ligase from Short Oligomers by Nonenzymatic Ligation.

Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.

Template-Directed Copying of RNA by Non-enzymatic Ligation.

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3'-amino group at the 3'-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.

A Model for the Emergence of RNA from a Prebiotically Plausible Mixture of Ribonucleotides, Arabinonucleotides, and 2'-Deoxynucleotides.

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

Nonenzymatic RNA-templated Synthesis of N3'→P5' Phosphoramidate DNA.

The RNA world hypothesis describes a scenario where early life forms relied on RNA to govern both inheritance and catalyze useful chemical reactions. Prior to the emergence of enzymes capable of replicating the RNA genome, a nonenzymatic replication process would have been necessary to initiate Darwinian Evolution. However, the one-pot nonenzymatic RNA chemical copying of templates with mixed-sequences is insufficient to generate strand products long enough to encode useful function. The use of alternate (RNA-like) genetic polymers may overcome hurdles associated with RNA copying, and further our understanding of nonenzymatic copying chemistry. This protocol describes the nonenzymatic copying of RNA templates into N3'→P5' phosphoramidate DNA (3'-NP-DNA). We describe, in detail, the synthesis of 3'-amino-2',3'-dideoxyribonucleotide monomers activated with 2-aminoimidazole (3'-NH2-2AIpddN), and their use in template-directed polymerization.

Clinical effects of epidurally administered dexmedetomidine with or without lidocaine in sheep.

BACKGROUND: The aims of this study were evaluate cardiopulmonary, sedative and antinociceptive effects of dexmedetomidine-lidocaine combination via lumbosacral epidural injection in sheep. METHODS: Six Santa Inês breed sheep, 16±6 months old and weighing 42.2 ± 5.7 kg were used. Sheep were subjected to epidural anaesthesia with three treatments: L, lidocaine (1.2 mg/kg), D, dexmedetomidine (2.5 µg/kg) or DL, dexmedetomidine plus lidocaine (2.5 µg/kg + 1.2 mg/kg). Drugs were injected via pre-placed lumbosacral epidural catheters. Cardiopulmonary, arterial blood gases, electrolytes, degree of sedation and antinociceptive aspects were measured before drug administration (T0) and then at 15, 30, 60 and 120 min after drug injection (T15-T120) in all treatments and at T0 to T240 in DL. RESULTS: There were significantly increases in PaCO2 at times T60 and T120 in D, and at T30-T120 in DL, compared to baseline. The antinociceptive effects were observed up to 240 min in DL and 60 min in L, and were more intense in DL. Treatment D provided analgesia only in the perineal region, and only at T15. CONCLUSION: The combination of DEX with lidocaine produced similar cardiopulmonary changes compared with either drug alone, but with greater and more prolonged antinociceptive effects.