Pilot evaluation of a virtual training program for child injury prevention.

Unintentional injuries are a leading cause of child death. The present study evaluated the effectiveness of a behavioral injury prevention program for children aged 3-18 years and their caregivers. To accommodate families during the Coronavirus-19 pandemic, training was modified to be delivered virtually. Forty-one children aged 3-18 years and 14 parents/caregivers of children aged 3-5 years attended one of several 4-hour online injury prevention training sessions directed toward residents of Washington state. Training was targeted to three different developmental stages (ages 3-5, 6-12 and 13-18 years). Study outcomes included knowledge about injury prevention strategies, perceived vulnerability for injury, self-efficacy to engage in safety behaviors and behavioral intentions to be safe. Following training, participants showed improved self-efficacy to stay safe, excellent knowledge about the learned material and increased behavioral intention to engage safely. There was minimal change in perceived vulnerability to injury among children; caregivers of young children felt their children were somewhat less vulnerable to injury following the training. Almost all participants said they would recommend the program to others. Results suggest that a virtual behavioral training program delivered remotely is feasible and may be effective to create behavior change and reduce child injury risk. Given its scalability and reach, such programs are recommended for further study, refinement and, if demonstrated effective in larger-scale controlled trials, dissemination to address the leading cause of child mortality in the United States, unintentional injury.

The structural biology of voltage-gated calcium channel function and regulation.

Voltage-gated calcium channels (CaVs) are large (approximately 0.5 MDa), multisubunit, macromolecular machines that control calcium entry into cells in response to membrane potential changes. These molecular switches play pivotal roles in cardiac action potentials, neurotransmitter release, muscle contraction, calcium-dependent gene transcription and synaptic transmission. CaVs possess self-regulatory mechanisms that permit them to change their behaviour in response to activity, including voltage-dependent inactivation, calcium-dependent inactivation and calcium-dependent facilitation. These processes arise from the concerted action of different channel domains with CaV beta-subunits and the soluble calcium sensor calmodulin. Until recently, nothing was known about the CaV structure at high resolution. Recent crystallographic work has revealed the first glimpses at the CaV molecular framework and set a new direction towards a detailed mechanistic understanding of CaV function.

Controlling potassium channel activities: Interplay between the membrane and intracellular factors.

Neural signaling is based on the regulated timing and extent of channel opening; therefore, it is important to understand how ion channels open and close in response to neurotransmitters and intracellular messengers. Here, we examine this question for potassium channels, an extraordinarily diverse group of ion channels. Voltage-gated potassium (Kv) channels control action-potential waveforms and neuronal firing patterns by opening and closing in response to membrane-potential changes. These effects can be strongly modulated by cytoplasmic factors such as kinases, phosphatases, and small GTPases. A Kv alpha subunit contains six transmembrane segments, including an intrinsic voltage sensor. In contrast, inwardly rectifying potassium (Kir) channels have just two transmembrane segments in each of its four pore-lining alpha subunits. A variety of intracellular second messengers mediate transmitter and metabolic regulation of Kir channels. For example, Kir3 (GIRK) channels open on binding to the G protein betagamma subunits, thereby mediating slow inhibitory postsynaptic potentials in the brain. Our structure-based functional analysis on the cytoplasmic N-terminal tetramerization domain T1 of the voltage-gated channel, Kv1.2, uncovered a new function for this domain, modulation of voltage gating, and suggested a possible means of communication between second messenger pathways and Kv channels. A yeast screen for active Kir3.2 channels subjected to random mutagenesis has identified residues in the transmembrane segments that are crucial for controlling the opening of Kir3.2 channels. The identification of structural elements involved in potassium channel gating in these systems highlights principles that may be important in the regulation of other types of channels.

Potassium channels: life in the post-structural world.

More than three years have passed since the first structure of a potassium channel protein revealed fundamental molecular details of a platform for ion-selective conduction. Recent efforts have turned to understanding what this structure tells us about potassium channel structure and function in general and, most importantly, which questions remain unanswered. Successes in solving membrane protein structures are still hard won and slow. High-resolution studies of cytoplasmic channel domains and channel-associated proteins, the most tractable entry points for dissecting large, complex eukaryotic channels, are revealing a modularity of function commonly seen in many other biological systems. Studies of these domains bring into sharp focus issues of channel regulation, how these domains and associated proteins are coupled to the transmembrane domains to influence channel function, and how ion channels are integrated into cellular signaling pathways.

The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel.

Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Recent mutagenesis of two T1 C-terminal loop residues implicates T1 in channel gating. However, structural alterations of these mutants leave open the question concerning direct involvement of T1 in gating. We find in mammalian Kv1.2 that gating depends critically on residues at complementary T1 surfaces in an unusually polar interface. An isosteric mutation in this interface causes surprisingly little structural alteration while stabilizing the closed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel. Together, these data suggest that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening.

Transmembrane structure of an inwardly rectifying potassium channel.

Inwardly rectifying potassium channels (K(ir)), comprising four subunits each with two transmembrane domains, M1 and M2, regulate many important physiological processes. We employed a yeast genetic screen to identify functional channels from libraries of K(ir) 2.1 containing mutagenized M1 or M2 domains. Patterns in the allowed sequences indicate that M1 and M2 are helices. Protein-lipid and protein-water interaction surfaces identified by the patterns were verified by sequence minimization experiments. Second-site suppressor analyses of helix packing indicate that the M2 pore-lining inner helices are surrounded by the M1 lipid-facing outer helices, arranged such that the M1 helices participate in subunit-subunit interactions. This arrangement is distinctly different from the structure of a bacterial potassium channel with the same topology and identifies helix-packing residues as hallmark sequences common to all K(ir) superfamily members.

Effects of nonfiber carbohydrate and niacin on periparturient metabolic status and lactation of dairy cows.

Fifty Holstein cows and 25 Holstein heifers were used in a randomized complete block design. Treatments were a standard nonfiber carbohydrate diet beginning at 19 d prepartum, a high nonfiber carbohydrate diet beginning at 19 d prepartum, a standard nonfiber carbohydrate diet plus niacin (12 g/d) beginning at 19 d prepartum, a high nonfiber carbohydrate diet plus niacin beginning at 19 d prepartum, and a standard nonfiber carbohydrate diet beginning at 19 d prepartum plus niacin beginning at 14 d postpartum. Treatments were applied through 40 wk postpartum. Niacin did not significantly affect production parameters or blood and liver metabolites. Prepartum intakes of dry matter and energy and energy balance were greater for cows and heifers fed the high nonfiber carbohydrate diets. Plasma glucose concentrations tended to be significantly higher, and nonesterified fatty acids and beta-hydroxybutyrate concentrations were significantly lower, when animals were fed diets that were high in nonfiber carbohydrates. Diets containing high nonfiber carbohydrates increased concentrations of liver glycogen and tended to reduce concentrations of liver triglyceride. Milk production tended to be higher, milk fat percentage tended to be lower, and milk protein percentage and production were significantly greater when diets that were high in nonfiber carbohydrates were fed. An increase in dietary nonfiber carbohydrates improved the metabolic parameters during the transition period and improved lactation performance.

Context-dependent secondary structure formation of a designed protein sequence.

Protein secondary structures have been viewed as fundamental building blocks for protein folding, structure and design. Previous studies indicate that the propensities of individual amino acids to form particular secondary structures are the result of a combination of local conformational preferences and non-local factors. To examine the extent to which non-local factors influence the formation of secondary structural elements, we have designed an 11-amino-acid sequence (dubbed the 'chameleon' sequence) that folds as an alpha-helix when in one position but as a beta-sheet when in another position of the primary sequence of the IgG-binding domain of protein G (GB1). Both proteins, chameleon-alpha and chameleon-beta, are folded into structures similar to native GB1, as judged by several biophysical criteria. Our results demonstrate that non-local interactions can determine the secondary structure of peptide sequences of substantial length. They also support views of protein folding that favour tertiary interactions as dominant determinants of structure.

Identification of D-peptide ligands through mirror-image phage display.

Genetically encoded libraries of peptides and oligonucleotides are well suited for the identification of ligands for many macromolecules. A major drawback of these techniques is that the resultant ligands are subject to degradation by naturally occurring enzymes. Here, a method is described that uses a biologically encoded library for the identification of D-peptide ligands, which should be resistant to proteolytic degradation. In this approach, a protein is synthesized in the D-amino acid configuration and used to select peptides from a phage display library expressing random L-amino acid peptides. For reasons of symmetry, the mirror images of these phage-displayed peptides interact with the target protein of the natural handedness. The value of this approach was demonstrated by the identification of a cyclic D-peptide that interacts with the Src homology 3 domain of c- SRC. Nuclear magnetic resonance studies indicate that the binding site for this D-peptide partially overlaps the site for the physiological ligands of this domain.

Using three-dimensional quantitative structure-activity relationships to examine estrogen receptor binding affinities of polychlorinated hydroxybiphenyls.

Certain phenyl-substituted hydrocarbons of environmental concern have the potential to disrupt the endocrine system of animals, apparently in association with their estrogenic properties. Competition with natural estrogens for the estrogen receptor is a possible mechanism by which such effects could occur. We used comparative molecular field analysis (CoMFA), a three-dimensional quantitative structure-activity relationship (QSAR) paradigm, to examine the underlying structural properties of ortho-chlorinated hydroxybiphenyl analogs known to bind to the estrogen receptor. The cross-validated and conventional statistical results indicate a high degree of internal predictability for the molecules included in the training data set. In addition to the phenolic (A) ring system, conformational restriction of the overall structure appears to play an important role in estrogen receptor binding affinity. Hydrophobic character as assessed using hydropathic interaction fields also contributes in a positive way to binding affinity. The CoMFA-derived QSARs may be useful in examining the estrogenic activity of a wider range of phenyl-substituted hydrocarbons of environmental concern.

Synthesis and molecular modeling of 1-phenyl-1,2,3,4-tetrahydroisoquinolines and related 5,6,8,9-tetrahydro-13bH-dibenzo[a,h]quinolizines as D1 dopamine antagonists.

New 1-phenyl-1,2,3,4-tetrahydroisoquinolines and related 5,6,8,9-tetrahydro- 13bH-dibenzo[a,h]-quinolizines were prepared as ring-contracted analogs of the prototypical 1-phenyl-2,3,4,5-tetrahydrobenzazepines (e.g., SCH23390) as a continuation of our studies to characterize the antagonist binding pharmacophore of the D1 dopamine receptor. Receptor affinity was assessed by competition for [3H]SCH23390 binding sites in rat striatal membranes. The 6-bromo-1-phenyltetrahydroisoquinoline analog 2 of SCH23390 1 had D1 binding affinity similar to that for the previously reported 6-chloro analog 6, whereas the 6,7-dihydroxy analog 5 had significantly lower D1 affinity. Conversely, neither 6-monohydroxy- (3) nor 7-monohydroxy-1-phenyltetrahydroisoquinolines (4) had significant affinity for the D1 receptor. These results demonstrate that 6-halo and 7-hydroxy substituents influence D1 binding affinity of the 1-phenyltetrahydroisoquinolines in a fashion similar to their effects on 1-phenyltetrahydrobenzazepines. The conformationally constrained 3-chloro-2-hydroxytetrahydrodibenzoquinolizine 9 had much lower affinity relative to the corresponding, and more flexible, 6-chloro-7-hydroxy-1-phenyltetrahydroisoquinoline 6. Similarly, 2,3-dihydroxytetrahydrodibenzoquinolizine 10 had much lower D1 affinity compared to dihydrexidine 14, a structurally similar hexahydrobenzo[a]phenanthridine that is a high-affinity full D1 agonist. Together, these data not only confirm the effects of the halo and hydroxy substitutents on the parent nucleus but demonstrate the pharmacophoric importance of both the nitrogen position and the orientation of the accessory phenyl ring in modulating D1 receptor affinity and function. Molecular modeling studies and conformational analyses were conducted using the data from these new analogs in combination with the data from compounds previously synthesized. The resulting geometries were used to refine a working model of the D1 antagonist pharmacophore using conventional quantitative structure-activity relationships and three-dimensional QSAR (CoMFA).

Context is a major determinant of beta-sheet propensity.

Residues in beta-sheets occur in two distinct tertiary contexts: central strands, bordered on both sides by other beta-strands, and edge strands, bordered on only a single side by another beta-strand. The delta delta G values for beta-sheet formation measured at an edge beta-strand of the IgG-binding domain of protein G(GB1) are quite different from those obtained previously at a central position in the same protein. In particular, there is no correlation at the edge position with statistically determined beta-sheet-forming preferences. The differences between beta-sheet propensities measured at central and edge beta-strands, delta delta delta G values, correlate with the values of water/octanol transfer free energies and side-chain non-polar surface area for the amino acids. These results strongly suggest that, unlike alpha-helix formation, beta-sheet formation is determined in large part by tertiary context, even at solvent-accessible sites, and not by intrinsic secondary structure preferences.

Measurement of the beta-sheet-forming propensities of amino acids.

Several model systems have been used to evaluate the alpha-helical propensities of different amino acids. In contrast, experimental quantitation of beta-sheet preferences has been addressed in only one model system, a zinc-finger peptide. Here we measure the relative propensity for beta-sheet formation of the twenty naturally occurring amino acids in a variant of the small, monomeric, beta-sheet-rich, IgG-binding domain from protein G. Amino-acid substitutions were made at a guest site on the solvent-exposed surface of the beta-sheet. Several criteria were used to establish that the mutations did not cause significant structural changes: binding to the Fc domain of IgG, calorimetric unfolding and NMR spectroscopy. Characterization of the terminal stabilities of these proteins leads to a thermodynamic scale for beta-sheet propensities that spans a range of approximately 2 kcal mol-1 for the naturally occurring amino acids, excluding proline. The magnitude of the differences suggests that beta-sheet preferences can be important determinants of protein stability.

Continuous interleukin-2 and tumor-infiltrating lymphocytes as treatment of advanced melanoma. A national biotherapy study group trial.

Melanoma metastases were harvested from 82 patients for the purpose of growing and expanding tumor-infiltrating lymphocytes (TIL). Tumor tissue cell suspensions were incubated with interleukin-2 (IL-2), followed by repeated exposure to tumor antigen with or without OKT3 monoclonal antibody (MoAb). Initial growth success was achieved in 56 of 82 cultures (72%). Efforts were made to expand 26 of these 56 cultures for therapeutic TIL; 23 of 26 early cultures (88%) were successfully expanded for in vivo therapy. It took a mean of 78.5 +/- 25.4 days to grow sufficient TIL for treatment. Therapy included cyclophosphamide (1 g/m2) on day 1, followed by a 96-hour continuous infusion of IL-2 (18 x 10(6) IU/m2/d) on days 2 to 5, and approximately 10(11) (mean 1.49 +/- 0.93 x 10(11)) TIL on day 2. Patients who responded received monthly IL-2 as a 96-hour infusion. Median patient age was 45 years of age. Sixty-seven percent of the patients were men. Performance status was 0 to 1 in 77% of patients. Thirty-four percent of the patients had liver metastases. The usual IL-2 toxicities were seen. Response rate for 21 patients was 24% (95% confidence interval, 10% to 49%). One complete response was achieved with cells 98% CD4+; four partial responses were achieved with cells 80%, 94%, 98%, and 98% CD8+, respectively. Four of eight patients who received TIL, which had never been stimulated with OKT3, had tumor response. The authors conclude that a treatment plan for IL-2/TIL is technically difficult, costly, and effective for only a minority of patients. Overall, clinical results are not clearly superior to those obtained with other IL-2 regimens.

Continuous infusion interleukin-2 and tumor-derived activated cells as treatment of advanced solid tumors: a National Biotherapy Study Group Trial.

Metastases from patients with solid tumors were harvested from 196 patients for the purpose of growing tumor-derived activated cells (TDAC). Cells were prepared from autologous tumor cultures by incubation with Interleukin-2 (IL-2) followed by repeated exposure to tumor antigen and/or anti-CD3 monoclonal antibody. Initial growth success was achieved in 66%; 45/56 (80%) of these early cultures were subsequently expanded for in vivo therapy. It took a mean of 69.4 +/- 24.0 days to grow TDAC for treatment. Thirty-eight patients were treated with cyclophosphamide (1 g/m2) on day one followed by a 96-hour continuous infusion of IL-2 (18 x 10(6) IU/m2/day) on days 2-5 and approximately 10(11) TDAC on day 2. Patients subsequently received monthly IL-2 as a 96-hour constant infusion if their cancers were stable or regressing. Median age was 51 yrs; 58% were male. Performance status was 0-1 in 64%, 29% had lung metastases; 34% had liver metastases. The usual IL-2 toxicities were seen. Responses were seen only in 1/38 patients (3%); a partial response in a patient with lymphoma. Forty-two percent were stable 90 days post-treatment, the rest were progressive or inevaluable. We conclude that a treatment plan for IL-2/TDAC is technically difficult, costly, and not practical under these conditions. Clinical results to date are not clearly different than those obtained with other IL-2 regimens.

A randomized, double-blind, placebo-controlled trial of transfer factor as adjuvant therapy for malignant melanoma.

One hundred and sixty-eight evaluable patients participated in a randomized, double-blind study of transfer factor (TF) versus placebo as surgical adjuvant therapy of Stage I and Stage II malignant melanoma. Eighty-five patients received TF prepared from the leukocytes of healthy volunteer donors; eighty-three participants received placebo. Therapy was initiated within 90 days of resection of all evident tumor and continued until 2 years of disease-free survival or the occurrence of unresectable dissemination of melanoma. Known prognostic variables were similarly distributed in the treatment and control groups, documenting the randomization efficacy. Three endpoints were analyzed: disease-free interval, time to Stage III metastasis, and survival. After a median follow-up period of 24.75 months, there was a trend in favor of the placebo group with regard to all three endpoints and this was significant (P less than or equal to 0.05) for time to Stage III metastasis. These findings indicate that TF is not effective as surgical adjuvant therapy of malignant melanoma.

A clinical and pharmacokinetic study of isolated limb perfusion with heat and melphalan for melanoma.

The pharmacokinetics of isolated limb perfusion were studied to see what melphalan concentrations were achieved and how effective the isolation was. Twenty-eight patients received 32 limb perfusions with heat and melphalan for locally recurrent or level V melanoma. Melphalan was given 0.75 mg/kg for axillary/popliteal or 1.2 mg/kg for femoral perfusions with heat (perfusate 42 degrees C, limb 40 degrees C) for 1 hour. Melphalan concentratives were measured by high-performance liquid chromatography in seven patients. Peak perfusate melphalan concentrations were 6.1 to 115 mg/ml, which was one to two logs higher than peak systemic concentratives of melphalan. Isolation of the perfusate circuit from the systemic circulation was better for axillary and popliteal perfusions than for femoral perfusions (P less than 0.05). Complete responses were seen in 81% of evaluable patients; long-term local control was achieved in most patients, although many developed hematogenous metastases. Toxicity included erythema and edema in all, mild leukopenia in two, neuropathy in two, and amputation was required in one patient. Improvements in surgical technique include regional anesthesia to reduce vasospasms and transcutaneous measurement of fluorescein to measure leak. Perfusion with heat and melphalan remains the treatment of choice for in-transit metastases from melanoma.

Metastatic islet cell tumor in von Hippel-Lindau disease.

A 56-year-old woman with many unusual manifestations of von Hippel-Lindau syndrome is described. In addition to retinal hemangioblastomas, pheochromocytoma, renal cell carcinoma, and multiple organ cysts, she had a cerebellar astrocytoma, pancreatic exocrine insufficiency, diabetes mellitus, thyrotoxicosis, and a metastatic calcitonin-secreting islet cell carcinoma. This case report documents the first example of a metastatic islet cell tumor in a patient with von Hippel-Lindau disease. The possible relationship between this disorder, the other neurocutaneous syndromes, and the multiple endocrine neoplasia syndromes is discussed.