Evaluation of reproducible and transparent research practices in pulmonology.

BACKGROUND: Study reproducibility is valuable for validating or refuting results. Provision of reproducibility indicators, such as materials, protocols, and raw data in a study improve its potential for reproduction. Efforts to reproduce noteworthy studies in the biomedical sciences have resulted in an overwhelming majority of them being found to be unreplicable, causing concern for the integrity of research in other fields, including medical specialties. Here, we analyzed the reproducibility of studies in the field of pulmonology. METHODS: 500 pulmonology articles were randomly selected from an initial PubMed search for data extraction. Two authors scoured these articles for reproducibility indicators including materials, protocols, raw data, analysis scripts, inclusion in systematic reviews, and citations by replication studies as well as other factors of research transparency including open accessibility, funding source and competing interest disclosures, and study preregistration. FINDINGS: Few publications included statements regarding materials (10%), protocols (1%), data (15%), and analysis script (0%) availability. Less than 10% indicated preregistration. More than half of the publications analyzed failed to provide a funding statement. Conversely, 63% of the publications were open access and 73% included a conflict of interest statement. INTERPRETATION: Overall, our study indicates pulmonology research is currently lacking in efforts to increase replicability. Future studies should focus on providing sufficient information regarding materials, protocols, raw data, and analysis scripts, among other indicators, for the sake of clinical decisions that depend on replicable or refutable results from the primary literature.

Discovery of a pathway for terminal-alkyne amino acid biosynthesis.

Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction1-4. However, biologically 'rare' functional groups, such as the fluoro5, chloro6,7, bromo7,8, phosphonate9, enediyne10,11, cyano12, diazo13, alkene14 and alkyne15-17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu(I)-catalysed azide-alkyne cycloaddition 'click' reaction18. Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces cattleya. We found that L-lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C-C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications.

Use of speech production repair strategies to improve diver communication.

The purpose of this investigation was to determine if speech intelligibility improved when divers made specific modifications to their speaking patterns while in a hyperbaric helium-oxygen (heliox) environment. Divers were trained to produce a variety of sentences using speech with three types of alterations: (1) slowed rate, (2) increased loudness, and (3) a combination of slightly slowed rate, a minimal increase in loudness, increased pause time, and greater mouth opening (composite strategy). Both diver and non-diver listeners judged these sentences for intelligibility. In addition, acoustic analysis of the cues for the identification of voicing, place, and manner of articulation was conducted to determine if such cues might become more audible in the speech signal when repair strategies were used. Both perceptual and acoustic results showed the composite method to be the best for natural-sounding, intelligible speech. It had the effect of slowing rate and increasing loudness just enough to increase intelligibility without causing distortion. It was concluded that teaching divers to produce speech using this method would be a worthwhile investment for improving speech intelligibility.

Phosphoaspartates in bacterial signal transduction.

Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.

A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad.

The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N'). This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme. The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol). The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32. It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled. The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin. Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin.

Father-child contact in inner-city African American families with maternal HIV infection.

This study examines father-child contact in inner-city African American families with maternal HIV infection. Participants were 246 African American women, 40% of whom are infected with HIV, and one of their non-infected children. Children from non-infected families were more likely to have fathers who are alive and who are living in the home. In addition, regardless of whether or not the father lived in the home, these children had more frequent father contact than children from families with maternal HIV infection. Explanations and implications of the findings are discussed.

Insight into schizophrenia: planning mental health service provision for schizophrenia using a brief psychological intervention.

Six nurses were trained to deliver an Insight programme to patients with schizophrenia and their main carers. One outcome of the intervention was the highlighting of recommendations for future treatment. These were fed back on an individual basis through an exit letter to the patient's consultant psychiatrist and key worker. The individual treatment recommendations were then collated into a report for each site and presented to clinical coordinators and business managers. It is envisaged that the recommendations could be used by managers in developing a business case for future mental health provision in schizophrenia. The complete results of the study are due to be published in the near future.

Crystal structure of activated CheY. Comparison with other activated receiver domains.

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.

Crystal structure of an activated response regulator bound to its target.

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

NMR structure of activated CheY.

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains.

Development and use of a virtual NMR facility.

We have developed a "virtual NMR facility" (VNMRF) to enhance access to the NMR spectrometers in Pacific Northwest National Laboratory's Environmental Molecular Sciences Laboratory (EMSL). We use the term virtual facility to describe a real NMR facility made accessible via the Internet. The VNMRF combines secure remote operation of the EMSL's NMR spectrometers over the Internet with real-time videoconferencing, remotely controlled laboratory cameras, real-time computer display sharing, a Web-based electronic laboratory notebook, and other capabilities. Remote VNMRF users can see and converse with EMSL researchers, directly and securely control the EMSL spectrometers, and collaboratively analyze results. A customized Electronic Laboratory Notebook allows interactive Web-based access to group notes, experimental parameters, proposed molecular structures, and other aspects of a research project. This paper describes our experience developing a VNMRF and details the specific capabilities available through the EMSL VNMRF. We show how the VNMRF has evolved during a test project and present an evaluation of its impact in the EMSL and its potential as a model for other scientific facilities. All Collaboratory software used in the VNMRF is freely available from www.emsl.pnl.gov:2080/docs/collab.

Spectroscopic methods for analysis of protein secondary structure.

Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.

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.

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators.

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.

Solution structure of the DNA-binding domain of NtrC with three alanine substitutions.

The structure of the 20 kDa C-terminal DNA-binding domain of NtrC from Salmonella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456, Asn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeric enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal domain contains the determinants necessary for dimerization and DNA-binding of the full length protein. The mutant protein does not bind to DNA but retains many characteristics of the wild-type protein, and the mutant domain expresses at high yield (20 mg/l) in minimal medium. Three-dimensional (1)H/(13)C/(15)N triple-resonance, (1)H-(13)C-(13)C-(1)H correlation and (15)N-separated nuclear Overhauser effect (NOE) spectroscopy experiments were used to make backbone and side-chain (1)H,(15)N, and (13)C assignments. The structures were calculated using a total of 1580 intra and inter-monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrogen bond restraints), and 88 phi dihedral restraints for residues Asp400 through Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were also included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) and B (Leu421-His440) join with those of another monomer to form an antiparallel four-helix bundle. Helices C (Gln446-Leu451) and D (Ala456-Met468) of each monomer adopt a classic helix-turn-helix DNA-binding fold at either end of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6 (+/-0.2) A. Structural differences between the C-terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed.

Success of cervical exploration for patients with asymptomatic primary hyperparathyroidism.

BACKGROUND: This study examined the success and safety of cervical exploration in patients with asymptomatic primary hyperparathyroidism compared with those with symptomatic disease. METHODS: Records of patients undergoing cervical exploration for primary hyperparathyroidism from June 1990 to October 1996 were reviewed. Patients were divided into three groups: (1) asymptomatic, (2) symptomatic, and (3) afflicted (those with associated complications). Information collected consisted of preoperative and postoperative symptoms, serum calcium and parathyroid hormone levels (PTH), and descriptions of pathologic and operative findings. RESULTS: Sixty-one patients were identified. Nineteen (31%) had no symptoms, 21 (34%) had subjective symptoms, and 21 had associated conditions, as described. Average preoperative and postoperative calcium levels were 11.5 mg% and 8.5 mg%, respectively. Average PTH levels also fell from 142 pg/mL to 49 pg/mL after surgery. Preoperative and postoperative calcium and PTH levels for the three groups showed no significant differences. The success of surgery in identifying pathology ranged from 90.5% to 95%, and again showed no difference among the three groups. Long-term morbidity (>6 months) in all groups was 0%. CONCLUSIONS: Cervical exploration and parathyroidectomy for asymptomatic primary hyperparathryoidism is safe and has similar success rates in identifying pathology and correcting biochemical abnormalities compared with patients with symptomatic disease.

Orphans of the AIDS epidemic in the United States: transition-related characteristics and psychosocial adjustment at 6 months after mother's death.

This study has two purposes: (1) to describe the characteristics related to the transition to orphanhood for children whose mothers die from AIDS and (2) to examine the psychosocial adjustment of these children at six months following maternal death. Twenty orphans and a control sample of 40 children from the same neighbourhoods, as well as their mothers or care-givers, served as participants. Two assessments occurred: (1) prior to the death of the mother in the orphan group and (2) six months after her death. The results indicated that relatives, particularly maternal grandparents, became the new care-giver of the orphans, no more than one residential move had occurred following the mother's death, and the new care-givers were providing a stable home environment. Child psychosocial adjustment did not change following maternal death.

Circumferential forearm fasciocutaneous free flap reconstruction of forequarter amputation/chest wall resection using simultaneous extra-anatomic revascularization (SEAR).

BACKGROUND: This report describes a technique in which temporary extra-anatomic revascularization of an amputated part was used to preserve a free flap while tumor resection and chest wall reconstruction were performed. METHODS: A patient with multiple local recurrences of basosquamous carcinoma of the shoulder underwent forequarter amputation with en bloc resection of the upper chest wall. During the resection, an elbow disarticulation of the amputated limb was performed. The vascular pedicle of the amputated forearm was joined to the dorsalis pedis vessels of the foot. Following completion of tumor resection and chest wall reconstruction, the forearm was disconnected from the foot and re-anastomosed to thoracic vessels, and a circumferential fasciocutaneous free flap was then harvested and inset. RESULTS: No ischemic flap complications occurred, and the patient recovered well. Ample time was afforded for complete tumor resection with negative margins and prosthetic reconstruction of the chest wall. CONCLUSIONS: The technique of temporary, simultaneous extra-anatomic revascularization of an amputated part for later free flap harvest may be helpful in avoiding potentially long flap ischemia times in selected complex oncologic resections.

Mapping of a protein-RNA kissing hairpin interface: Rom and Tar-Tar*.

An RNA 'kissing' complex is formed by the association of two hairpins via base pairing of their complementary loops. This sense-antisense RNA motif is used in the regulation of many cellular processes, including Escherichia coli ColE1 plasmid copy number. The RNA one modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to the kissing hairpins and stabilizing their interaction. We have used heteronuclear two-dimensional NMR spectroscopy to map the interface between Rom and a kissing complex formed by the loop of the trans -activation response (Tar) element of immunodeficiency virus 1 (HIV-1) and its complement. The protein binding interface was obtained from changes in amide proton signals of uniformly 15N-labeled Rom with increasing concentrations of unlabeled Tar-Tar*. Similarly, the RNA-binding interface was obtained from changes in imino proton signals of uniformly 15N-labeled Tar with increasing concentrations of unlabeled Rom. Our results are in agreement with previous mutagenesis studies and provide additional information on Rom residues involved in RNA binding. The kissing hairpin interface with Rom leads to a model in which the protein contacts the minor groove of the loop-loop helix and, to a lesser extent, the major groove of the stems.

Speech intelligibility assessment in a helium environment. II. The speech intelligibility index.

The Speech Intelligibility Index (SII) was measured for Navy divers participating in two saturation deep dives and for a group of nondivers to test different communication systems and their components. These SIIs were validated using the Speech Perception in Noise (SPIN) test and the Griffiths version of the Modified Rhyme Test (GMRT). Our goal was to determine if either of these assessments was sensitive enough to provide an objective measure of speech intelligibility when speech was processed through different helmets and helium speech unscramblers (HSUs). Results indicated that SII values and percent intelligibility decreased incrementally as background noise level increased. SIIs were very reliable across the different groups of subjects indicating that the SII was a strong measurement for predicting speech intelligibility to compare linear system components such as helmets. The SII was not useful in measuring intelligibility through nonlinear devices such as HSUs. The speech intelligibility scores on the GMRT and SPIN tests were useful when the system component being compared had a large measurable difference, such as in helmet type. However, when the differences were more subtle, such as differences in HSUs, neither the SPIN nor the GMRT appeared sensitive enough to make such distinctions. These results have theoretical as well as practical value for measuring the quality and intelligibility of helium speech enhancement systems.