Considerations for the design of clinical trials for tinnitus

CONCLUSION: There are many possible control conditions to consider in designing research on tinnitus treatments. Some of the counseling procedures involve more than simply 'talking' or providing information, and it is important to make this distinction. Several good handicap scales are available, but we believe that 100-point interval scales have some superior attributes. Both primary and secondary measures of benefit should be used. Open trials have some merit, but should only be used cautiously. Several recent guidelines have been suggested for improving the design and reporting of clinical trials. OBJECTIVES: This paper reviews some basic considerations in the design of research to evaluate tinnitus treatments, particularly counseling and sound therapies. METHODS: We have reviewed some of the basic issues, referenced some relevant work, and provided some data supporting some of our assertions. RESULTS: We provide some recommendations for consideration for the design of clinical trials.     

A simulation based technique to estimate intracluster correlation for a binary variable

Cluster randomized trials have become the design of choice for evaluating the effect of selected interventions on well-known health indicators such as neonatal mortality rate, episiotomy rate, and postpartum hemorrhage rate in a community setting. Determining the sample size of a cluster randomized trial requires a reliable estimate of cluster size and the intracluster correlation (ICC), because sample size can be substantially impacted by these parameters. During the design phase of a trial, the investigators may have estimates of the valid range of the health indicator which is the primary outcome variable. Furthermore, investigators often have an estimate of the average cluster size or range of cluster sizes that exist among the proposed samples they are planning to include in the trial. We present in this article a simulation technique to estimate the ICC value and its distribution for known binary outcome variables and a varying number of clusters and cluster sizes. We applied this technique to estimate ICC values and confidence intervals for a multi-country trial assessing the effect of neonatal resuscitation to decrease seven-day neonatal mortality, where communities within a country were clusters. This simulation technique can be used to estimate the possible ranges of the ICC values and to help to design an appropriately powered trial.     

<Superscript>31</Superscript>P cardiac magnetic resonance spectroscopy during leg exercise at 3 Tesla

Investigation of phosphorus (³¹P) magnetic resonance spectroscopy under stress conditions provides a non-invasive tool to examine alterations in cardiac high-energy phosphate metabolism that may not be evident at rest. Our aim was to establish cardiac ³¹P MR spectroscopy during leg exercise at 3T. The increased field strength should provide a higher signal to noise ratio than at lower field strengths. Furthermore, relatively high temporal resolution at a sufficiently fine spatial resolution should be feasible. ³¹P MR spectra were obtained with a 3D acquisition weighted chemical shift imaging sequence in 20 healthy volunteers at rest, during dynamic physiological leg exercise and after recovery at 3T. Haemodynamic measurements were made throughout and the rate pressure product calculated. With exercise, the mean heart rate increased by 73%, achieving a mean increase in rate pressure product of 115%. The corrected PCr/ATP ratio for subjects at rest was 2.02 ± 0.43, exercise 2.14 ± 0.67 (P = 0.54 vs. rest) and at recovery 2.03 ± 0.52 (P = 0.91 vs. rest, P = 0.62 vs. exercise). A cardiac ³¹P MR spectroscopy physiological exercise-recovery protocol is feasible at 3T. There was no significant change in high-energy cardiac phosphate metabolite concentrations in healthy volunteers at rest, during physiological leg exercise or during recovery. When applied to patients with heart disease, this protocol should provide insights into physiological and pathological cardiac metabolism.     

Artificial voice modulation in dogs by recurrent laryngeal nerve stimulation: electrophysiological confirmation of anatomic data

OBJECTIVES: We hypothesized that voice may be artificially manipulated to ameliorate dystonias considered to be a failure in dynamic integration between competing neuromuscular systems. METHODS: Orderly intrinsic laryngeal muscle recruitment by anodal block via the recurrent laryngeal and vagus nerves has allowed us to define specific values based on differential excitabilities, but has precluded voice fluency because of focused breaks during stimulation and the need to treat several neural conduits. Such problems may be obviated by a circuit capable of stimulating some axons while simultaneously blocking others in the recurrent laryngeal nerve, which carries innervation to all intrinsic laryngeal muscles, including the arguably intrinsic cricothyroideus. In 5 dogs, both recurrent laryngeal nerves received 40-Hz quasi-trapezoidal pulses (0 to 2000 microA, 0 to 2000 micros, 0 to 500 micros decay) via tripolar electrodes. Electromyograms were matched with audio intensities and fundamental frequencies recorded under a constant flow of humidified air. Data were digitized and evaluated for potential correlations. RESULTS: Orderly recruitment of the thyroarytenoideus, posterior cricoarytenoideus, and cricothyroideus was correlated with stimulating intensities (p < .001), and posterior cricoarytenoideus opposition to the thyroarytenoideus and cricothyroideus was instrumental in manipulating audio intensities and fundamental frequencies. CONCLUSIONS: Manipulation of canine voice parameters appears feasible via the sole recurrent laryngeal nerve within appropriate stimulation envelopes, and offers promise in human laryngeal dystonias.     

Physiology and phenomenology of tinnitus: implications for treatment

We examine a contrast in understanding tinnitus and how this impacts on treatment approaches. First, a physiological account of tinnitus is described based on disinhibition and cortical remapping following injury at the receptor level, the analog for tinnitus being the 'phantom limb pain' phenomenon. Secondly, an experimental model of tinnitus is reviewed that relies on inference from conditioning animal behaviour. Arising from this, a role for conditioning in people distressed by tinnitus has been proposed, based on the unfounded premise that, for humans, tinnitus is a neutral stimulus, the distress being due to association with other stressful events. We critique this because we believe it influences approaches to tinnitus treatment. Finally, the phenomenology of tinnitus in the human case is analysed, with its nature illuminated via a series of distinctions with hearing impairment. Tinnitus can be intrinsically stressful for some people. Understanding this emphasizes the need to involve concepts and treatment in the area of clinical psychology. A flexible coalition between clinical audiologists and clinical psychologists is proposed as fruitful for tinnitus and related rehabilitation.     

Establishing a tinnitus clinic in your practice

PURPOSE: While tinnitus is very common among the hearing impaired population, specific treatment for tinnitus is not provided in most clinics. This article provides a plan for establishing a tinnitus treatment program that can be implemented in stages at most audiology clinics. METHOD: Preparation for establishing a tinnitus clinic includes having an overall plan regarding the type and degree of tinnitus management. Assessment involves a measurement of tinnitus and of the reaction a patient has to the tinnitus, including the use of handicap questionnaires. Management typically involves some form of counseling and sound therapy. Four problematic areas in tinnitus management are thoughts and emotions, hearing and communication, sleep, and concentration. CONCLUSIONS: Licensed audiologists generally have the essential training necessary to provide counseling and sound therapy to treat tinnitus patients. We introduce 3 levels of treatment implementation, depending on whether the patient is curious, concerned, or distressed. Follow-up and referrals might be necessary in more severe cases. Finally, the development of a tinnitus clinic centers around establishing a need for individual treatment, creating a treatment plan, estimating the need for additional staff and resources, reimbursement options, and assessing the effectiveness of the program.     

Expression of the type III TGF-beta receptor is negatively regulated by TGF-beta

The type III transforming growth factor-beta receptor (TbetaRIII or betaglycan) is a ubiquitously expressed transforming growth factor-beta (TGF-beta) superfamily coreceptor with essential roles in embryonic development. Recent studies have defined a role for TbetaRIII in the pathogenesis of human cancers, with frequent loss of TbetaRIII expression at the message and protein level. Mechanisms for the loss of TbetaRIII expression remain to be fully defined. Advanced human cancers often have elevated circulating levels of TGF-beta1. Here, we define a specific role for TGF-beta1 in negatively regulating TbetaRIII at the message level in breast and ovarian cancer models. TGF-beta1 decreased TbetaRIII message and protein levels in ovarian (Ovca420) and breast cancer (MDA-MB-231) cell lines in both a dose- and time-dependent manner. TGF-beta1-mediated TbetaRIII repression is mediated by the type I TGF-beta receptor/Smad2/3 pathway as the activin receptor-like kinase 5 (ALK5) inhibitor, SB431542, abrogated this effect, while the expression of constitutively active ALK5 was sufficient to repress TbetaRIII expression. Mechanistically, TGF-beta1 does not affect TbetaRIII messenger RNA (mRNA) stability, but instead directly regulates the TbetaRIII promoter. We define alternative promoters for the TGFBR3 gene, a distal and proximal promoter. Although both promoters are active, only the proximal promoter was responsive and negatively regulated by TGF-beta1 and constitutively active ALK5. Taken together, these studies define TGF-beta1-mediated downregulation of TbetaRIII mRNA expression through effects on the ALK5/Smad2/3 pathway on the TGFBR3 gene proximal promoter as a potential mechanism for decreased TbetaRIII expression in human cancers.     

Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein–protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.

Glycosylation of asparagine residues is one of the most abundant and structurally complex protein posttranslational modifications (1, 2) and occurs in all domains of life (3). Owing to their relatively large size and hydrophilicity or simply their presence at definite locations, asparagine-linked (N-linked) glycans can significantly alter protein properties including biological activity, chemical solubility, folding and stability, immunogenicity, and serum half-life (4, 5). Hence, glycosylation effectively increases the diversity of the proteome by enriching the repertoire of protein characteristics beyond that dictated by the 20 canonical amino acids. For example, accumulating evidence indicates that the immune system diversifies the repertoire of antigen specificities by exclusively targeting the antigen-binding sites of immunoglobulins (IgGs) with posttranslational modifications, in particular N-linked glycosylation (6). Moreover, the profound effect of glycans on proteins has prompted widespread glycoengineering efforts to rationally manipulate key glycosylation parameters (e.g., glycan size and structural composition and glycosite location and occupancy) as a means to optimize protein traits for a range of different industrial and therapeutic applications (7–10).Despite some notable successes, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is currently limited by our inability to predict which sites within a protein are glycosylatable and how glycosylation at permissive sites will affect protein structure and function. Indeed, a deeper understanding of the design rules (i.e., how glycans influence the biological and biophysical properties of a protein) represents a grand challenge for the glycoprotein engineering field. To this end, computational approaches have enabled in silico exploration of glycosylation-induced effects on protein folding and stability (11, 12); however, these involve a trade-off between molecular detail and glycoprotein size, with full-atomistic molecular dynamics simulations typically limited to only short glycopeptides or protein domains (11). To experimentally probe the consequences of glycosylation ideally requires access to large collections of chemically defined glycoproteins in sufficient quantities for characterization (13). Mammalian cells represent an obvious choice to source proteins with both natural and naïve glycosites. However, because of the time-consuming, low-throughput nature of gene transfection and culturing of mammalian cells, studies using mammalian cell-based expression systems have typically only investigated a small number of designs (∼15 or fewer) (14–17), with rare exceptions such as the tour de force study by Elliott et al. (18). In addition, the intrinsic variability with respect to the glycan structure at a given site (microheterogeneity) can be unpredictable and difficult to control in mammalian expression systems. Another option is chemical synthesis, which can furnish structurally uniform glycopeptides for investigating the local effects of N-linked glycans on peptide conformation (19). While total chemical synthesis remains challenging for full-length proteins, advances in expressed protein ligation (EPL) have opened the door to convergent assembly of chemically synthesized glycopeptides with recombinantly expressed protein domains to form larger glycoproteins bearing complex N-glycans installed at discrete sites (20, 21). Using this technology, Imperiali and coworkers created a panel of seven site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the disaccharide N,N''-diacetylchitobiose (GlcNAc₂) and assessed the kinetic and thermodynamic consequences of glycan installation at defined locations (22). Unfortunately, EPL is a technically demanding procedure, requiring manual construction of each individual glycoprotein, which effectively limits the number of testable glycosite designs to just a small handful.To move beyond these “one-glycosite-at-a-time” methods for supplying glycoproteins, herein we describe a scalable technique called shotgun scanning glycomutagenesis (SSGM) that involves design and construction of combinatorial acceptor protein libraries in which 1) each member of the library carries a single N-glycosite “mutation” introduced at a defined position along the protein backbone and 2) the complete ensemble of glycan acceptor sites (sequons) in the library effectively covers every possible position in the target protein (Fig. 1). The resulting SSGM libraries are expressed using N-glycosylation-competent bacteria in the context of glycoSNAP (glycosylation of secreted N-linked acceptor proteins), a versatile high-throughput screen based on extracellular secretion of glycosylated proteins (23). Using this glycoprotein engineering tool, we constructed and screened SSGM libraries corresponding to three model proteins: bacterial immunity protein Im7, bovine pancreatic ribonuclease A (RNase A), and a human single-chain variable fragment antibody specific for HER2 (scFv-HER2). Our results revealed that installation of N-glycans was tolerated at a large number of positions and in all types of secondary structure, with relatively high N-glycosylation efficiency in the majority of cases. For many of these glycoproteins, the presence of N-glycans at naïve sites had a measurable effect on protein stability and/or activity in a manner that depended on the precise location of the modification. Taken together, these findings demonstrate the ability of the SSGM method to yield large collections of discretely modified neoglycoproteins that collectively reveal glycosylatable sites and provide insight on the influence that site-specific N-glycan installation has on structural and/or functional properties.Open in a separate windowFig. 1.Constructing neoglycoproteins by SSGM. Schematic of SSGM, a glycoprotein engineering method based on combinatorial protein libraries in which glycosylation “sequon walking” is used to introduce an acceptor site at every possible position along a protein backbone. Note that the multiresidue nature of a sequon (e.g., N-X-S/T or D/E-X₁-N-X₂-S/T, where X, X₁, X₂ ≠ P) necessitates insertion or replacement of up to five additional amino acid substitutions at each position. The resulting library is expressed in glycoengineered bacteria, providing an opportunity for each library member to be expressed and glycosylated in a manner that is compatible with high-throughput screening via glycoSNAP to interrogate the glycosylation phenotype of individual variants. By integrating expressed SSGM libraries with multiplexable assays, the biochemical and biophysical properties of each neoglycoprotein can be individually interrogated. Depicted is the engineered C. jejuni GalNAc₅(Glc)GlcNAc heptasaccharide with reducing end GlcNAc (blue square) followed by five GalNAc residues (yellow squares) and a branching glucose (blue circle). Structure drawn according to symbol nomenclature for glycans (SNFG; https://www.ncbi.nlm.nih.gov/glycans/snfg.html).     

Hypothermia for the treatment of neonatal ischemic encephalopathy: is the genie out of the bottle?

Evidence suggests that hypothermia for hypoxic ischemic encephalopathy in the term neonate may decrease the risk of death or neurodevelopmental impairment. The objective of this study was to determine how hypothermia has been incorporated into practice. An anonymous survey was sent to medical directors of United States neonatal intensive care units (NICUs) in October 2005. We received completed surveys from 441 (54.5%) of 809 of NICUs. Only 6.4% of respondents used hypothermia. The most common method was total body cooling (64.3%) compared with head cooling (25%) or both (10.7%). At centers that did not offer hypothermia, 29% transferred infants to an institution that did. Centers that offered hypothermia were more likely at academic institutions (76.9%) compared with private practices (11.5%; p < 0.001). Hypothermia was more likely offered at institutions that offered extracorporeal membrane oxygenation (ECMO; 57%) than centers where ECMO was not offered (43%; p < 0.001). There has not been widespread use of hypothermia. There are a variety of protocols used. As results of further outcome studies become available, educational efforts and national practice guidelines will be essential.