Hierarchies of evidence applied to lifestyle Medicine (HEALM): introduction of a strength-of-evidence approach based on a methodological systematic review.

BACKGROUND: Current methods for assessing strength of evidence prioritize the contributions of randomized controlled trials (RCTs). The objective of this study was to characterize strength of evidence (SOE) tools in recent use, identify their application to lifestyle interventions for improved longevity, vitality, or successful aging, and to assess implications of the findings. METHODS: The search strategy was created in PubMed and modified as needed for four additional databases: Embase, AnthropologyPlus, PsycINFO, and Ageline, supplemented by manual searching. Systematic reviews and meta-analyses of intervention trials or observational studies relevant to lifestyle intervention were included if they used a specified SOE tool. Data was collected for each SOE tool. Conditions necessary for assigning the highest SOE grading and treatment of prospective cohort studies within each SOE rating framework were summarized. The expert panel convened to discuss the implications of findings for assessing evidence in the domain of lifestyle medicine. RESULTS AND CONCLUSIONS: A total of 15 unique tools were identified. Ten were tools developed and used by governmental agencies or other equivalent professional bodies and were applicable in a variety of settings. Of these 10, four require consistent results from RCTs of high quality to award the highest rating of evidence. Most SOE tools include prospective cohort studies only to note their secondary contribution to overall SOE as compared to RCTs. We developed a new construct, Hierarchies of Evidence Applied to Lifestyle Medicine (HEALM), to illustrate the feasibility of a tool based on the specific contributions of diverse research methods to understanding lifetime effects of health behaviors. Assessment of evidence relevant to lifestyle medicine requires a potential adaptation of SOE approaches when outcomes and/or exposures obviate exclusive or preferential reliance on RCTs. This systematic review was registered with the International Prospective Register of Systematic Reviews, PROSPERO [CRD42018082148].

Variations in the presence of chloride cells in the gills of lampreys (Petromyzontiformes) and their evolutionary implications.

Although confined to fresh water, non-parasitic species of lampreys and the landlocked parasitic sea lamprey, all of which were derived relatively recently from an adromous ancestors, still develop chloride cells, whose function in their ancestors was for osmoregulation in marine waters during the adult parasitic phase. In contrast, such cells are not developed by the non-parasitic least brook lamprey Lampetra aepyptera, which has been separated from its ancestor for >2 million years, nor by the freshwater parasitic species of the genus Ichthyomyzon. The length of time that a non-parasitic species or landlocked parasitic form or species has spent in fresh water is thus considered the overriding factor determining whether chloride cells are developed by those lampreys.

Examining new phylogenetic markers to uncover the evolutionary history of early-diverging fungi: comparing MCM7, TSR1 and rRNA genes for single- and multi-gene analyses of the Kickxellomycotina.

The recently recognised protein-coding genes MCM7 and TSR1 have shown significant promise for phylogenetic resolution within the Ascomycota and Basidiomycota, but have remained unexamined within other fungal groups (except for Mucorales). We designed and tested primers to amplify these genes across early-diverging fungal clades, with emphasis on the Kickxellomycotina, zygomycetous fungi with characteristic flared septal walls forming pores with lenticular plugs. Phylogenetic tree resolution and congruence with MCM7 and TSR1 were compared against those inferred with nuclear small (SSU) and large subunit (LSU) rRNA genes. We also combined MCM7 and TSR1 data with the rDNA data to create 3- and 4-gene trees of the Kickxellomycotina that help to resolve evolutionary relationships among and within the core clades of this subphylum. Phylogenetic inference suggests that Barbatospora, Orphella, Ramicandelaber and Spiromyces may represent unique lineages. It is suggested that these markers may be more broadly useful for phylogenetic studies among other groups of early-diverging fungi.

Importance of side marking in ophthalmic surgery.

BACKGROUND: In 2003 the National Patient Safety Agency (NPSA) advised side marking for avoiding errors and reducing incorrect side surgery. This survey aimed to ascertain whether or not, these 'best practice' guidelines are currently being implemented by ophthalmic surgeons in Scotland and, if not, the reasons for this, and also to ascertain surgeons' attitudes towards marking. METHODS: Semi-structured interviews with ophthalmic surgeons throughout Scotland, either face to face or by telephone, on their current practices and attitudes relating to preventing wrong side surgery. RESULTS: Non-compliance with side marking was described by 48% of the surgeons, which includes both consultants and specialist registrars. This survey reveals incomplete implementation of this policy in ophthalmic surgery for a number of reasons. The most common was bypassing the established multi-step process of checks, which risks an increased likelihood of surgical errors. CONCLUSION: Guidelines are not consistently being implemented in their entirety by eye surgeons in Scotland. In order to improve compliance and improve patient safety, we suggest a risk-stratified approach in side marking based on individual patient factors that may encourage wider acceptance, without compromising patient safety.

Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features.

Charybdotoxin (CTX), a 37 amino acid protein isolated from the venom of L. quinquestriatus, is a high-affinity blocker of various Ca2(+)-activated K+ channels. CTX also blocks Drosophila Shaker (Sh) clone H4 transient K+ currents expressed in Xenopus oocytes with similar affinity (Kd = 3.6 nM). CTX blocks both the open and the closed states of Sh channels with no apparent change in gating behavior. In addition, the block is enhanced as the ionic strength is lowered. These properties are identical to those of CTX block of Ca(+)-activated K+ channels, and these results suggest that the external pore openings of these two functionally dissimilar K+ channels may share common structural features.

Activation of squid axon K+ channels. Ionic and gating current studies.

We have used data obtained from measurements of ionic and gating currents to study the process of K+ channel activation in squid giant axons. A marked improvement in the recording of K+ channel gating currents (IKg) was obtained by total replacement of Cl- in the external solution by NO-3, which eliminates approximately 50% of the Na+ channel gating current with no effect on IKg. The midpoint of the steady state charge-voltage (Qrel - V) relationship is approximately 40 mV hyperpolarized to that of the steady state activation (fo - V) curve, which is an indication that the channel has many nonconducting states. Ionic and gating currents have similar time constants for both ON and OFF pulses. This eliminates any Hodgkin-Huxley nx scheme for K+ channel activation. An interrupted pulse paradigm shows that the last step in the activation process is not rate limiting. IKg shows a nonartifactual rising phase, which indicates that the first step is either the slowest step in the activation sequence or is voltage independent. These data are consistent with the following general scheme for K+ channel activation: (formula; see text)

Probes of the conduction process of a voltage-gated Cl- channel from Torpedo electroplax.

The open-channel conductance properties of a voltage-gated Cl- channel derived from Torpedo californica electroplax and incorporated into planar bilayers were studied by several approaches. In neutral bilayers the channel conductance saturates with Cl- activity according to a rectangular hyperbolic relation with a half-saturation activity of 75 mM and a maximum conductance of 32 pmho. The observation of identical behavior in charged membranes implies that ions permeating the channel do not sense the surface potential of the bulk membrane. The Cl-:Br- permeability ratio, measured under biionic conditions, is independent of salt concentration. SCN- ion reversibly blocks the channel. The voltage dependence of the block implies the existence of two separate blocking sites within the channel: one accessible from the cis side only (the side to which vesicles are added) and the other accessible from the trans side only. The block at each site is competitive with Cl-. The results are consistent with a single-ion Eyring model of the conduction process in which the ion must traverse three kinetic barriers as it permeates the channel and in which the channel can accommodate at most one ion at a time.

Adolescent smoking decline during California's tobacco control programme.

OBJECTIVE: California's comprehensive tobacco control programme was 13 years old in 2002; by then, children entering adolescence at the start of the programme were young adults. This study examines whether adolescent smoking declined over this period, whether any decline carried through to young adulthood, and whether it was specific to California. SETTING AND PARTICIPANTS: Most data were from the 1990-2002 California Tobacco Surveys (CTS) (adolescents 12-17 years, > 5000/survey, young adults 18-24 years, > 1000/survey). Additional data were from the national 1992/93-2001/02 Current Population Survey (CPS) (young adults 18-24 years, > 15,000/survey). RESULTS: Over the 13 year period in California, ever puffing declined by 70% in 12-13 year olds, by 53% in 14-15 year olds from 1992-2002, and by 34% in 16-17 year olds from 1996-2002 (CTS). As noted, the decline commenced progressively later in each older group. Smoking experimentation (1+ cigarettes) and established smoking (> 100 cigarettes in lifetime) showed similar patterns. Compared to 1990, the percentage of California young adults (CTS data) who ever experimented declined by 14%, with half of the decline from 1999-2002. CPS young adult smoking prevalence (established and now smoke everyday or some days) was constant in the rest of the USA over the entire period, but California showed a recent 18% decline from 1998/99 to 2001/02. CONCLUSIONS: California's comprehensive programme may have kept new adolescent cohorts from experimenting with cigarettes. Low young adolescent experimentation rates at programme start appeared to carry through to young adulthood, resulting in a recent drop in young adult smoking prevalence in California not observed in the rest of the USA.

Ligand-receptor interactions in the nicotinic acetylcholine receptor probed using multiple substitutions at conserved tyrosines on the alpha subunit.

Affinity labeling studies have identified several conserved tyrosine residues in the alpha subunit of the nicotinic acetylcholine receptor (alpha Y93, alpha Y190, and alpha Y198) as being in or near the ligand binding site. Mutagenesis studies from several laboratories have shown that substitution of phenylalanine for tyrosine at these positions reduces the apparent affinity for ACh. We have examined this apparent reduction in affinity further through the use of multiple substitutions at each position. Substitution of either phenylalanine, tryptophan, or serine resulted in an apparent decrease in agonist affinity, but the degree of reduction depended on both the position and the nature of the substitution. Analysis of the effects of each substitution suggests that each residue interacts with the quaternary N of ACh, and that each residue may make a different type of interaction with the agonist.

Role of in vitro cholesterol depletion in mediating human platelet aggregation.

We investigated the direct role of cholesterol lowering on human platelet aggregation by in vitro cholesterol depletion using methyl-beta-cyclodextrin. Collagen and thrombin receptor agonist peptide induced maximal aggregation was significantly decreased in cholesterol depleted platelets. In contrast, anti-CD9 antibody, mAb7, or anti-beta(3) antibody, D3, induced percent maximal aggregation was unaffected by cholesterol depletion. Surface and total alpha(IIb)beta(3) levels were equivalent in both groups. Morphological and ultrastructural analysis of collagen induced aggregates revealed that normal and cholesterol depleted platelets changed shape and aggregated; however, cholesterol depletion impaired microtubule ring formation and aggregate size. Cholesterol depletion also diminished the extent of the open canalicular system and collagen induced platelet ATP release. These data suggest cholesterol depletion impairs platelet aggregation by altering platelet ultrastructure critical in mediating secretion. Temporal differences and differences in tyrosine phosphoprotein levels following collagen stimulation were observed, thereby indicating that platelet signaling was concurrently affected by cholesterol depletion.

Interaction of D-tubocurarine analogs with the 5HT3 receptor.

D-Tubocurarine is a potent competitive antagonist of two members of the ligand-gated ion channel family, the muscle-type nicotinic acetylcholine receptor (AChR) and serotonin type-3 receptor (5HT3R). We have used a series of analogs of D-tubocurarine to determine the effects of methylation, stereoisomerization and halogenation on the interaction of D-tubocurarine with the 5HT3R. The affinities of the analogs for the 5HT3R span a 200-fold concentration range and fall into three broad groups. The first group, with affinity constants (Ki) < 150 nM, consists of D-tubocurarine and analogs modified at the nitrogens or 7' hydroxyl. The fact that these compounds all have high affinity for the 5HT3R suggests that these portions of the ligand do not make interactions with the receptor that are critical for high-affinity binding. The second group, with Ki's in the 1-5 microM range, consists of analogs modified at the 12'-hydroxyl or the adjacent 13'-carbon, which suggests that this portion of the ligand makes interactions that are important for high-affinity binding. The third, very low affinity, group is a compound with altered stereoconfiguration at the 1 carbon, demonstrating the importance of proper configuration of the antagonist in ligand-receptor interactions. For the most part, this pattern of selectivity is similar to that for the AChR, suggesting that the structures of the ligand-binding sites of these two receptors share common structural features.

Interspecies comparison of platelet aggregation, LIBS expression and clot retraction: observed differences in GPIIb-IIIa functional activity.

We examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species' platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

Assessment of lumiaggregometry for research and clinical laboratories.

Platelet aggregometry is often used to help diagnose storage pool disease (SPD-reduced amounts of granule nucleotides) and release defects (abnormal release of granule nucleotides). The general assumption that normal aggregation patterns are sufficient to rule out the diagnosis of one of these disorders has been invalidated by the recent publication of two papers describing patients with clinical bleeding, prolonged bleeding times and normal aggregation patterns in spite of defective release. The lumiaggregometer provides a tool for measuring platelet release and aggregation simultaneously. This paper presents a standardized, reproducible method for the use of the lumiaggregometer based on a "standard curve". Data obtained during the development of the procedure are presented including normal ranges of release at different concentrations of agonists, release measured in intrinsic disorders as well as in patients on aspirin, and values for release relative to varying platelet counts. A monoclonal antibody (anti-p24/CD9; MAb7) which activates platelets similarly to thrombin and may be a useful reagent for distinguishing SPD and release defects is also introduced.

A Ser162-->Leu mutation within glycoprotein (GP) IIIa (integrin beta3) results in an unstable alphaIIbbeta3 complex that retains partial function in a novel form of type II Glanzmann thrombasthenia.

Platelets from Glanzmann thrombasthenia patient BL express approximately 30% of the normal alphaIIbbeta3 content and support fibrin-mediated clot retraction, but fail to bind fibrinogen or aggregate following cellular activation. BL platelets bind neither activation-dependent nor activation-independent ligands. DNA sequence analysis of BL platelet mRNA revealed a homozygous C583-->T point mutation in a conserved region of beta3, resulting in a Ser162Leu amino acid substitution. This mutation appears to produce destabilizing effects on the alphaIIbbeta3 complex, as evidenced by the fact that (1) the BL alphaIIbbeta3 complex exhibited altered sedimentation velocity through sucrose gradients, (2) alphaIIb and beta3 was not recognized by complex-dependent monoclonal antibodies or co-precipitated by integrin subunit-specific antibodies, and (3) biosynthesis and trafficking of the alphaIIbbeta3Leu162 complex was delayed relative to that of the wild-type control. Taken together, these data implicate the region encompassing Ser162 in the stabilization and ligand binding properties of the alphaIIbbeta3 complex.

Effects of pregnancy and chronic hypoxia on contractile responsiveness to alpha1-adrenergic stimulation.

Decreased contractile response to vasoconstrictors in uterine and nonuterine vessels contributes to increased blood flow to the uterine circulation during normal pregnancy. Pregnancies complicated by preeclampsia and/or chronic hypoxia show a reversal or diminution of these pregnancy-associated changes. We sought to determine whether chronic hypoxia opposes the reduction in contractile response in uterine and nonuterine vessels during normal pregnancy and, if so, whether decreased basal nitric oxide (NO) activity was involved. We examined the contractile response to phenylephrine (PE) in guinea pig uterine artery (UA), mesenteric artery (MA), and thoracic aorta (TA) rings isolated from nonpregnant or pregnant guinea pigs that had been exposed throughout gestation to either low (1,600 m, n = 47) or high (3,962 m, n = 43) altitude. In the UA, pregnancy reduced contractile sensitivity to PE and did so similarly at low and high altitude (EC50: 4.0 x 10(-8) nonpregnant, 9.3 x 10(-8) pregnant at low altitude; 4.8 x 10(-8) nonpregnant, 1.0 x10(-8) pregnant at high altitude; both P < 0.05). Addition of the NO synthase inhibitor nitro-L-arginine (NLA; 200 mM) to the vessel bath increased contractile sensitivity in the pregnant UA (P < 0.05) and eliminated the effect of pregnancy at both altitutes. NLA also raised contractile sensitivity in the nonpregnant high-altitude UA, but contractile response without NLA did not differ in the high- and low-altitude animals. In the MA, pregnancy decreased contractile sensitivity to PE at high altitude only, and this shift was reversed by NO inhibition. In the TA, neither pregnancy nor altitude affected contractile response, but NO inhibition raised contractile response in nonpregnant and pregnant TA at both altitudes. We concluded that pregnancy diminished contractile response to PE in the UA, likely as a result of increased NO activity, and that these changes were similar at low and high altitude. Counter to our hypothesis, chronic hypoxia did not diminish the pregnancy-associated reduction in contractile sensitivity to PE or inhibit basal NO activity in the UA; rather it enhanced, not diminished, basal NO activity in the nonpregnant UA and the pregnant MA.

Does cigarette price influence adolescent experimentation?

The economics literature generally agrees that state and federal excise taxes can play an important role in deterring adolescent smoking. Teens' apparent responsiveness to cigarette prices is puzzling, since the majority of adolescent smokers do not buy their cigarettes. Teens typically do not begin to purchase cigarettes until they have developed an established pattern of smoking. Previous studies have not had adequate measures of smoking experience to explore whether adolescents' price responsiveness may vary by smoking experience. This paper uses data from a 1993 national survey of youth smoking to explore this hypothesis.

Dose response of topical carbamylcholine chloride (carbachol) in normotensive and early glaucomatous beagles.

Single-dose loading with a placebo (0.5% methylcellulose) and 0.75%, 1.5%, 2.25%, or 3% carbamylcholine chloride (carbachol) was evaluated in normotensive and glaucomatous Beagles to determine dose response. The 4 carbachol concentrations significantly (P less than 0.05) lowered intraocular pressure and reduced pupil size in the normotensive and early glaucomatous Beagles at most time intervals as compared with base-line values, nontreated fellow eyes, and placebo-treated eyes. There were no significant (P less than 0.24) effects on intraocular pressure or pupil size by unilateral carbachol instillations as compared with base-line values or placebo-treated eyes.

Effects of clomipramine hydrochloride on dominance-related aggression in dogs.

OBJECTIVE: To compare effects of the serotonergic drug clomipramine hydrochloride with those of placebo for treatment of dominance-related aggression in dogs. DESIGN: Randomized, placebo-controlled, double-blind clinical trial. ANIMALS: 28 neutered dogs > 1 year old with dominance-related aggression. PROCEDURE: Dogs displaying > or = 3 aggressive episodes/wk toward > or = 1 human family member in response to identifiable behavioral triggers were included in the study. Owners were instructed not to change patterns of interaction with their dogs during the study. After 2 weeks of baseline observations, dogs were treated for 6 weeks with clomipramine (1.5 mg/kg [0.7 mg/lb] of body weight, q 12 h; n = 15) or placebo (13). Responses to triggers were assigned the following aggression scores: no response, 0; growl or lip curl, 1; snap or bite, 2. Mean scores for responses to triggers were obtained during the 2-week pretreatment period (baseline) and during the first and second weeks, third and fourth weeks, and fifth and sixth weeks of treatment. At the end of the study, owners assigned a score designed to evaluate their overall perceived change in aggressiveness; this was referred to as the global score. RESULTS: Mean aggression scores decreased at the fifth and sixth week of treatment in both groups, compared with baseline scores. However, mean scores between groups were not different. Global scores, assigned by the owner, generally reflected changes in mean aggression scores. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with placebo, clomipramine administered to dogs at the dosage recommended for treatment of separation anxiety did not reduce aggressiveness toward human family members.