Patient-reported outcomes from STARTRK-2: a global phase II basket study of entrectinib for ROS1 fusion-positive non-small-cell lung cancer and NTRK fusion-positive solid tumours.

BACKGROUND: Patient-reported outcomes (PROs) are increasingly relevant endpoints in clinical trials, contributing to our understanding of risk-benefit profiles, in addition to efficacy and safety data. We investigated the impact of entrectinib on patient-reported symptoms, functioning, and health-related quality of life. PATIENTS AND METHODS: STARTRK-2 is a phase II basket study in patients with locally advanced/metastatic neurotrophic receptor tyrosine kinase 1/2/3 (NTRK1/2/3) and ROS proto-oncogene 1 (ROS1) fusion-positive solid tumours. PROs (prespecified secondary endpoint) were evaluated using the European Organization for Research and Treatment of Cancer quality-of-life questionnaire (QLQ-C30), lung cancer module (QLQ-LC13), and colorectal cancer module (QLQ-CR29), and the EuroQoL 5-Dimension 3-Level instruments, completed before cycle 1 day 1 and each subsequent 4-week cycle of entrectinib dosing, and the end of treatment. Adverse events and treatment-related symptoms were assessed in the safety analysis (SA)-PRO population. Tumour-related symptoms, functioning, and global health status were assessed in the efficacy analysis (EA)-PRO population. Data cut-offs: 31 October 2018 NTRK cohort; 01 May 2019 ROS1 cohort. RESULTS: SA-PRO populations comprised patients with NTRK fusion-positive solid tumours (N = 88) or ROS1 fusion-positive non-small-cell lung cancer (N = 180) who received one or more doses of entrectinib, completed PRO questionnaires on cycle 1 day 1 and answered one or more questions on-study. EA-PRO populations (N = 71) and (N = 145), respectively, comprised SA-PRO patients with measurable baseline disease. Moderate-to-high baseline global health status scores were maintained in EA-PRO populations during treatment. Role and physical functioning scores were moderate-to-high at baseline, with trends towards clinical improvement during treatment. Both cohorts reported low-to-moderate symptom burden at baseline, which was maintained or trended towards clinically meaningful improvement. Symptoms commonly associated with cancer treatment (e.g. nausea, fatigue) remained stable or improved during treatment. All SA-PRO patients experienced one or more adverse events, most frequently constipation or diarrhoea. CONCLUSIONS: PRO findings were consistent with the favourable safety profile of entrectinib, and further reinforce the positive benefit-risk profile of this treatment, indicating minimal overall treatment burden.

A prospective observational study of needle-handling practices at a University Veterinary Teaching Hospital.

AIM: To determine the period prevalence of needlestick injury (NSI) at the Massey University Veterinary Teaching Hospital (VTH) and to identify handling and disposal practices that may contribute to the risk of NSI. METHODS: Observations of personnel were conducted in the equine (EVH) and companion animal (CAH) clinics of the VTH during scheduled clinical activities over 9- and 10-day periods, respectively. The number and type of NSI incidents, needle uncapping, capping and disposal events were recorded for veterinarians, nurses and other personnel (visitors and students). The number of needle-related practices, as a proportion of observations, were compared between CAH and EVH, and veterinarians, nurses and others using χ(2) tests. RESULTS: Needlestick injury was not observed during 190 and 163 needle handling and disposal observations in the CAH and EVH, respectively. Uncapping of needles by mouth was observed and was practised more by veterinarians (15/119; 13%) than nurses (2/42; 5%) and others (6/193; 3%) (p=0.001). Two-handed needle recapping after use was observed 265/354 times, and the one handed scooping technique was rarely observed (8/352). In the case of needle disposal, EVH workers used a container that was not purpose built for disposal more than CAH staff (p=0.02), or placed them in a pocket more frequently (p=0.003). Needle disposal containers were available on adjacent bench tops for 65/190 (34%) CAH observations, but no EVH observations. For 51/163 (31%) EVH observations the needle disposal containers were located on the ground, whereas none were observed there in the CAH. No approved sharps containers were observed in the immediate EVH and CAH work areas for 47/163 (28.8%) and 1/191 (0.5%) needle-handling activities, respectively. CONCLUSIONS: Unsafe needle-handling practices must be reduced by policies and training programmes to encourage safe needle-related practices, and ensuring that approved sharps containers are available in close proximity to where needles are used.

Towards an understanding of the control of 'crumbly' fruit in red raspberry.

The genetic disorder known as 'crumbly' fruit is becoming a serious problem in the European raspberry industry. The study set out to examine the crumbly phenotype in a red raspberry mapping population under two environments (field and polytunnel) across six seasons in an effort to understand variability of the syndrome and to examine whether genetic factors were important and if so, whether QTL associated with the phenotype could be identified. This highlighted that seasonal, environmental (field or polytunnel) and genetic factors all influence the condition. Two QTL that are important for the genetic control of the condition have been located on linkage groups one and three, and an association with ripening time has been identified.

CT chest abdomen pelvis doses in Scotland: has the DRL had its day?

OBJECTIVE: This article reports on a pilot study designed to collect dose data representative of current CT chest abdomen pelvis (CAP) practice in Scotland, make any immediately obvious interventions and to identify if the current UK diagnostic reference level (DRL) of 940 mGy cm is still appropriate. The aims are to identify if a Scotland-wide picture archiving and communication system (PACS)-based dose audit of a number of CT examinations is likely to have value in terms of optimization of patient doses and to comment on the significance of the results in terms of future optimization strategies. METHODS: Dose audit of CT CAP examinations at 32 different scanner sites across Scotland using accepted data collection and analysis methods. The minimum sample size was 30. RESULTS: RESULTS indicate that CT CAP doses are lower than those previously reported (median, 800 mGy cm, 75th percentile 840 mGy cm) but follow a distribution that is not in keeping with the concept of DRLs as presently understood or implemented. CONCLUSION: There is value in a PACS-based dose audit project to provide serial snapshots of patient doses as optimization efforts take place and to revise current knowledge about CT doses. In our opinion, the results call into question whether DRLs or the concept of "achievable dose" are suitable for devising optimization strategies once a certain degree of optimization has taken place. ADVANCES IN KNOWLEDGE: The results reported here suggest that it may be time to take a different approach to optimization, concentrating on tools that are more refined than the DRL, which may have become more of a compliance tool than an aid to optimization.

Inhibition of the mammalian target of rapamycin pathway by rapamycin blocks cocaine-induced locomotor sensitization.

Repeated cocaine exposure induces locomotor sensitization, which is mediated by adaptive changes in synaptic transmission in the mesolimbic dopamine pathway. The molecular mechanisms underlying this adaptation remain poorly understood. One pathway that may play a role is the mammalian target of rapamycin (mTOR) which is implicated in synaptic plasticity. In the present study, we found that cocaine exposure stimulates mTOR activity in rat brain. Furthermore, inhibition of mTOR by rapamycin blocked the induction as well as the expression of cocaine-induced locomotor sensitization in rats. These data elucidate a novel mechanism by which the mTOR pathway mediates cocaine-induced behavioral changes and could suggest a new interventional strategy for drug abuse.

Clay as thermoluminescence dosemeter in diagnostic radiology applications.

BACKGROUND: As part of efforts to isolate and utilize local and naturally occurring materials for development of thermoluminescece dosemeters and other technologies, an earlier report had shown that Nigerian clay showed prospects of utility as a thermoluminescence dosemeter (TLD). This paper reports the investigation of the basic thermoluminescence properties of clay at x-rays in the diagnostic radiology range, including dose monitoring in abdominal radiography. METHODOLOGY: Clay sourced from Calabar, Nigeria, was tested for thermoluminescence response after irradiation at diagnostic radiology doses, including application in abdominal radiography dose monitoring in a clinical setting. RESULTS: Results show that thermoluminescence (TL) output in natural clay is very low, but demonstrates enhanced performance with the addition of common salt. Specific TL characteristics of good repeatability for individual and batched pellets (variability index of 3.08%) and a high degree of trap emptying were observed. It had a glow curve peak at 275 degrees C; with traces of spurious thermoluminescence emission at the reader anneal temperature. There was evidence of good batch homogeneity (< 30%) and a similar pattern of dose absorption in abdominal radiography with commercial Lithium Fluoride (LiF TLD-100). A high fading rate (over 30% in twelve hours) and low sensitivity (12 times less than LiF TLD-100) however, signal the unacceptability of clay as a TLD in diagnostic radiology in the forms studied. CONCLUSION: Clay demonstrates poor TL response at diagnostic radiology doses. However, it's water absorbing property offers a means of overcoming the hygroscopic nature of common salt. This could be explored to improve the use of sodium chloride as a radiation detector.

Improvement in duration of erection following phosphodiesterase type 5 inhibitor therapy with vardenafil in men with erectile dysfunction: the ENDURANCE study.

OBJECTIVE: The ENDURANCE study evaluated the efficacy of vardenafil, a phosphodiesterase type 5 (PDE5) inhibitor, in men with erectile dysfunction (ED), by measuring the duration of erection leading to successful intercourse using a stopwatch as the assessment instrument. METHODS: This was a randomised, multicentre, double-blind, placebo-controlled, crossover study consisting of a 4-week treatment-free run-in phase after which patients were randomised to either fixed-dose vardenafil 10 mg or placebo (to be administered 60 min prior to intercourse) and entered the first of the two 4-week double-blind treatment periods, separated by a 1-week washout. The primary efficacy end-point was the stopwatch-assessed duration of erection, which was defined as the time from erection perceived hard enough for penetration until withdrawal from the partner's vagina leading to successful intercourse as measured by Sexual Encounter Profile Question 3 (SEP-3). Secondary efficacy end-points included SEP-2 and SEP-3 success rates, the erectile function domain of the International Index of Erectile Function, global assessment questionnaire, change from baseline in duration of erection and duration of erection not leading to successful intercourse. Safety was assessed by adverse events (AEs), laboratory samples, vital signs and ECGs. RESULTS: Of the 191 men included in the safety population, 40% had moderate ED and 33% had severe ED at baseline. The duration of erection (least squares mean +/- SE) leading to successful intercourse was longer with vardenafil than with placebo (12.81 +/- 1.00 min vs. 5.45 +/- 1.00 min; p < 0.001). The differences recorded for all secondary end-points were statistically significant in favour of vardenafil compared with placebo (p < 0.001), with the exception of duration of erection not leading to successful intercourse. Vardenafil was well tolerated in this study; the majority of AEs being mild-to-moderate in intensity. CONCLUSION: Vardenafil 10-mg therapy provided a statistically superior duration of erection leading to successful intercourse in men with ED compared with placebo.

Compensation in pre-synaptic dopaminergic function following nigrostriatal damage in primates.

Clinical symptoms of Parkinson's disease only become evident after 70-80% reductions in striatal dopamine. To investigate the importance of pre-synaptic dopaminergic mechanisms in this compensation, we determined the effect of nigrostriatal damage on dopaminergic markers and function in primates. MPTP treatment resulted in a graded dopamine loss with moderate to severe declines in ventromedial striatum (approximately 60-95%) and the greatest reductions (approximately 95-99%) in dorsolateral striatum. A somewhat less severe pattern of loss was observed for striatal nicotinic receptor, tyrosine hydroxylase and vesicular monoamine transporter expression. Declines in striatal dopamine uptake and transporter sites were also less severe than the reduction in dopamine levels, with enhanced dopamine turnover in the dorsolateral striatum after lesioning. The greatest degree of adaptation occurred for nicotine-evoked [(3)H]dopamine release from striatal synaptosomes, which was relatively intact in ventromedial striatum after lesioning, despite > 50% declines in dopamine. This maintenance of evoked release was not due to compensatory alterations in nicotinic receptor characteristics. Rather, there appeared to be a generalized preservation of release processes in ventromedial striatum, with K(+)-evoked release also near control levels after lesioning. These combined compensatory mechanisms help explain the finding that Parkinson's disease symptomatology develops only with major losses of striatal dopamine.

A patient with tremors and breathlessness.

We describe a patient with Parkinson's disease who developed bilateral pleural effusions and pleural fibrosis associated with pergolide therapy. Pergolide is an ergot-derived dopamine agonist used in the treatment of Parkinson's disease. This case report illustrates that physicians should have a high index of suspicion and consider drug-induced adverse effects in any differential diagnosis.

Spread of an epidemic Pseudomonas aeruginosa strain from a patient with cystic fibrosis (CF) to non-CF relatives.

Colonisation with Pseudomonas aeruginosa is common in adults with cystic fibrosis (CF) and there is increasing evidence that transmissible strains may cross colonise patients. However, transmission of these strains by social contact to healthy non-CF individuals has not been described. A case is presented where an adult CF patient colonised by an epidemic P aeruginosa strain infected her parents with subsequent morbidity.

The making of a gradient: IcsA (VirG) polarity in Shigella flexneri.

The generation and maintenance of subcellular organization in bacteria is critical for many cell processes and properties, including growth, structural integrity and, in pathogens, virulence. Here, we investigate the mechanisms by which the virulence protein IcsA (VirG) is distributed on the bacterial surface to promote efficient transmission of the bacterium Shigella flexneri from one host cell to another. The outer membrane protein IcsA recruits host factors that result in actin filament nucleation and, when concentrated at one bacterial pole, promote unidirectional actin-based motility of the pathogen. We show here that the focused polar gradient of IcsA is generated by its delivery exclusively to one pole followed by lateral diffusion through the outer membrane. The resulting gradient can be modified by altering the composition of the outer membrane either genetically or pharmacologically. The gradient can be reshaped further by the action of the protease IcsP (SopA), whose activity we show to be near uniform on the bacterial surface. Further, we report polar delivery of IcsA in Escherichia coli and Yersinia pseudotuberculosis, suggesting that the mechanism for polar delivery of some outer membrane proteins is conserved across species and that the virulence function of IcsA capitalizes on a more global mechanism for subcellular organization.

Superinfection with a transmissible strain of Pseudomonas aeruginosa in adults with cystic fibrosis chronically colonised by P aeruginosa.

Infection with transmissible strains of Pseudomonas aeruginosa can occur in uncolonised patients, but cross infection (superinfection) of patients already colonised withP aeruginosa has not been reported. With genotypic identification, we found superinfection by a multiresistant transmissible strain of P aeruginosa in four patients with cystic fibrosis (CF) who were already colonised by unique strains of P aeruginosa. No evidence of environmental contamination was found, but all patients became superinfected after contact with colonised individuals during inpatient stays. Inpatients with CF who are colonised with P aeruginosa should be separated by strain type. Such strain typing can only be reliably done by genomic methods, but this has resource implications.

Continuous-wave magnetic resonance imaging of short T(2) materials.

There is growing interest in the use of magnetic resonance imaging (MRI) to examine solid materials where the restricted motion of the probed spins leads to broad lines and short T(2) values, rendering many interesting systems invisible to conventional 2DFT pulsed imaging methods. In EPR T(2) seldom exceeds 0.1 mus and continuous-wave methods are adopted for spectroscopy and imaging. In this paper we demonstrate the use of continuous-wave MRI to obtain 2-dimensional images of short T(2) samples. The prototype system can image samples up to 50 mm in diameter by 60 mm long and has been used to image polymers and water penetration in porous media. Typical acquisition times range between 10 and 40 min. Resolution of 1 to 2 mm has been achieved for samples with T(2) values ranging from 38 to 750 mus. There is the possibility of producing image contrast that is determined by the material properties of the sample.

Nicotine self-administration in rats: estrous cycle effects, sex differences and nicotinic receptor binding.

RATIONALE: Research on smoking behavior and responsiveness to nicotine suggests that nicotine's effects may depend on the sex of the organism. OBJECTIVE: The present study addressed four questions: 1) Will female rats self-administer nicotine? 2) Does self-administration by females vary as a function of estrous cycle? 3) Does self-administration by females differ from that of males? 4) Does self-administration of nicotine result in up-regulation of nicotinic receptor binding and are these changes similar in males and females? METHODS: Male and female Sprague-Dawley rats were allowed to self-administer nicotine at one of four doses (0.02-0.09 mg/kg, free base) on both fixed and progressive ratio schedules of reinforcement. RESULTS: Females acquired nicotine self-administration across the entire range of doses. Acquisition of self-administration at the lowest dose was faster in females than males. However, few sex differences were found in the number of active responses, number of infusions, or total intake of nicotine during stable fixed ratio self-administration. In contrast, females reached higher break points on a progressive ratio. For both schedules, females had shorter latencies to earn their first infusion of each session and demonstrated higher rates of both inactive and timeout responding. There was no effect of estrous cycle on self-administration during either fixed or progressive ratio sessions. Self-administered nicotine resulted in average arterial plasma nicotine levels between 53 and 193 ng/ml and left hemi-brain levels between 174 and 655 ng/g, depending on dose. Nicotine self-administration produced similar up-regulation of nicotinic receptor binding sites in males and females, as reflected by increased right hemi-brain binding of [3H]-epibatidine, when compared to the brains of untreated control rats. CONCLUSIONS: These results suggest that while males and females may regulate their intake of nicotine similarly under limited access conditions, the motivation to obtain nicotine is higher in females.

Ligand-induced changes in the structure and dynamics of a human class Mu glutathione S-transferase.

Glutathione transferases are detoxification enzymes that catalyze the addition of glutathione (GSH) to a wide variety of hydrophobic compounds. Although this group of enzymes has been extensively characterized by crystallographic studies, little is known about their dynamic properties. This study investigates the role of protein dynamics in the mechanism of a human class mu enzyme (GSTM2-2) by characterizing the motional properties of the unliganded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these ligands are 10-20-fold lower than the diffusion limit, suggesting dynamic conformational heterogeneity of the active site. The off-rate of GSDNB is similar to the turnover number for its enzymatic formation, suggesting that product release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substrate. The dynamic properties of GSTM2-2 were investigated over a wide range of time scales using (15)N nuclear spin relaxation, residual dipolar couplings, and amide hydrogen-deuterium exchange rates. These data show that the majority of the protein backbone is rigid on the nanosecond to picosecond time scale for all forms of the enzyme. The presence of motion on the millisecond to microsecond time scale was detected for a small number of residues within the active site. These motions are likely to play a role in facilitating substrate binding and product release. The residual dipolar couplings also show that the conformation of the active site region is more open in solution than in the crystalline environment, further enhancing ligand accessibility to the active site. Amide hydrogen-deuterium exchange rates indicate a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate of a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site region. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 complex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic residues in the active site, including Met108. Replacement of Met108 with Ala increases the turnover number of the enzyme by a factor of 1.7.

Mecamylamine prevents tolerance but enhances whole brain [3H]epibatidine binding in response to repeated nicotine administration in rats.

RATIONALE: Chronic administration of nicotine in rats results in upregulation of neuronal nicotinic receptors. Upregulation has been proposed to reflect receptor desensitization, which may underlie functional tolerance to nicotine's effects. However, evidence indicates that tolerance and upregulation do not always parallel each other, suggesting that either upregulation does not always reflect desensitization, or mechanisms other than receptor desensitization account for tolerance to nicotine. OBJECTIVES: The present studies examined tolerance to nicotine-induced antinociception and changes in receptor binding after two regimens of intermittent nicotine injections in rats. The role of receptor activation in upregulation and tolerance was also examined by co-administering nicotine with the non-competitive antagonist, mecamylamine. METHODS: Sprague-Dawley rats were administered a short (once-daily, s.c. for 6 days (0.35 mg/kg)) or long (twice-daily for 11 days (0.66 mg/kg)) series of injections and tolerance to nicotine-induced antinociception and [3H]epibatidine binding in whole brain were measured. RESULTS: The short series of injections resulted in tolerance to nicotine-induced antinociception, but failed to increase [3H]epibatidine binding. In contrast, the long series of injections resulted in both tolerance and increased receptor binding. Once-daily pairings of mecamylamine (1 mg/kg, s.c.) with nicotine (0.35 mg/kg) for 6 days blocked the development of tolerance, indicating receptor activation is necessary for tolerance to occur. Pairing mecamylamine with nicotine (0.66 mg/kg) twice daily for 11 days blocked tolerance but produced a greater increase in [3H]epibatidine binding than nicotine alone. CONCLUSIONS: A dissociation of tolerance from receptor upregulation was observed in the present study. The finding that receptor activation may be necessary for tolerance but not upregulation is discussed within the context of possible mechanisms controlling tolerance to nicotine.

The discriminative stimulus properties of the atypical antipsychotic olanzapine in rats.

RATIONALE: Analysis of the preclinical behavioral effects of atypical antipsychotic agents will provide a better understanding of how they differ from typical antipsychotics and aid in the development of future atypical antipsychotic drugs. OBJECTIVES: The present study was designed to provide information about the discriminative stimulus properties of the atypical antipsychotic olanzapine. METHODS: Rats were trained to discriminate the atypical antipsychotic olanzapine (either 0.5 mg/kg OLZ or 0.25 mg/kg OLZ, i.p.) from vehicle in a twolever drug discrimination procedure. The atypical antipsychotic clozapine fully substituted for olanzapine in both the 0.5-mg/kg OLZ group (99.3% drug lever responding [DLR]) and the 0.25-mg/kg OLZ group (99.9% DLR). The typical antipsychotic chlorpromazine also substituted for olanzapine in both the 0.5-mg/kg OLZ group (87.5% DLR) and in the 0.25-mg/kg OLZ group (98.9% DLR); whereas, haloperidol displayed partial substitution for olanzapine in the 0.5-mg/kg OLZ group (56.1% DLR) and in the 0.25-mg/kg OLZ group (76.4% DLR). The 5.0-mg/kg dose of thioridazine produced olanzapine-appropriate responding in the 0.5-mg/kg OLZ group (99.6% DLR), but only partial substitution was seen with the 0.25-mg/kg OLZ training dose (64.0% DLR). The atypical antipsychotics raclopride (53.9% DLR) and risperidone (60.1% DLR) displayed only partial substitution in the 0.5-mg/kg OLZ group. Both the muscarinic cholinergic antagonist scopolamine (90.0% DLR) and the 5-HT2A/2C serotonergic antagonist ritanserin (86.0% DLR) fully substituted for olanzapine in the 0.5-mg/kg OLZ group. CONCLUSIONS: In contrast to previous discrimination studies with clozapine-trained rats, the typical antipsychotic agents chlorpromazine and thioridazine and the serotonin antagonist ritanserin substituted for olanzapine. These results demonstrate that there are differences in the mechanisms underlying the discriminative stimulus properties of clozapine and olanzapine. Specifically, olanzapine's discriminative stimulus properties appear to be meditated in part by both cholinergic and serotonergic mechanisms.

Nicotine self-administration in rats on a progressive ratio schedule of reinforcement.

RATIONALE: Robust intravenous (i.v.) nicotine self-administration (SA) in rats has been reported by several laboratories, including our own, using fixed ratio (FR) schedules of reinforcement. Studies on other drugs of abuse, however, suggest that progressive ratio (PR) schedules may provide additional information not gained using FR schedules. OBJECTIVE: Here, we attempt to establish and characterize nicotine SA on a PR. METHODS: One study allowed animals to acquire SA on a FR at four doses of nicotine (0.02, 0.03, 0.06, 0. 09 mg/kg) before being switched to a PR. A second study examined extinction by saline substitution or pretreatment with the nicotinic antagonist, mecamylamine, including a preliminary analysis into the role of secondary reinforcers in the extinction process. RESULTS: SA of nicotine on a PR was stable across repeated sessions. The number of infusions earned on a PR correlated with infusion rate on a FR; however, a large portion of the variance in SA on a PR could not be accounted for by infusion rate on a FR. Infusions on a PR increased across the same range of doses that produced a decrease in the infusion rate on a FR. Extinction of responding occurred after saline substitution or pretreatment with mecamylamine, and animals re-acquired when nicotine was again available without pretreatment. The presence of drug-paired stimuli appeared to lengthen the extinction process. CONCLUSIONS: Nicotine supports stable SA on a PR. Since PR and FR schedules may measure different aspects of nicotine reinforcement, PR schedules may be valuable in further characterizing group and individual differences in nicotine reinforcement.

A J(CH)-modulated 2D (HACACO)NH pulse scheme for quantitative measurement of (13)C(alpha)-(1)H(alpha) couplings in (15)N, (13)C-labeled proteins.

A triple-resonance pulse sequence is presented for the quantitative measurement of (1)H(alpha)-(13)C(alpha) single-bond couplings in (15)N, (13)C uniformly labeled proteins. This (1)J(CH)-modulated (HACACO)NH experiment yields (1)H(N)-(15)N-correlated 2D spectra in which the amplitude of each peak is modulated by the (1)H(alpha)-(13)C(alpha) J coupling of the preceding residue, (i - 1). The experiment is demonstrated on a 1.0 mM sample of Rho130, the 15-kDa RNA binding domain of E. coli Rho factor. The average error in the measured coupling constants was less than 0.8%. This sequence allows the measurement of the (1)J(CH )couplings from a proton-nitrogen HSQC without the need for assigning the H(alpha) and C(alpha) resonances.