Effectiveness of a home-based balance-training program in reducing sports-related injuries among healthy adolescents: a cluster randomized controlled trial

Background

Sport is the leading cause of injury requiring medical attention among adolescents. We studied the effectiveness of a home-based balance-training program using a wobble board in improving static and dynamic balance and reducing sports-related injuries among healthy adolescents.

Methods

In this cluster randomized controlled trial, we randomly selected 10 of 15 high schools in Calgary to participate in the fall of 2001. We then recruited students from physical education classes and randomly assigned them, by school, to either the intervention (n = 66) or the control (n = 61) group. Students in the intervention group participated in a daily 6-week and then a weekly 6-month home-based balance-training program using a wobble board. Students at the control schools received testing only. The primary outcome measures were timed static and dynamic balance, 20-m shuttle run and vertical jump, which were measured at baseline and biweekly for 6 weeks. Self-reported injury data were collected over the 6-month follow-up period.

Results

At 6 weeks, improvements in static and dynamic balance were observed in the intervention group but not in the control group (difference in static balance 20.7 seconds, 95% confidence interval [CI] 10.8 to 30.6 seconds; difference in dynamic balance 2.3 seconds, 95% CI 0.7 to 4.0 seconds). There was evidence of a protective effect of balance training in over 6 months (relative risk of injury 0.2, 95% CI 0.05 to 0.88). The number needed to treat to avoid 1 injury over 6 months was 8 (95% CI 4 to 35).

Interpretation

Balance training using a wobble board is effective in improving static and dynamic balance and reducing sports-related injuries among healthy adolescents.Adolescents commonly participate in sports.^1,2 In a survey of adolescents in Alberta, 59% reported that they took part in sports more than 5 hours per week (unpublished data). In North America, sport is the leading cause of injury requiring medical attention and visits to an emergency department among adolescents.^3,4 In Alberta 26% of youths aged 15–19 years in a survey reported sustaining a sports-related injury requiring medical attention.⁵ The impact may be lifelong, as there is evidence that knee and ankle injuries may result in an increased risk of osteoarthritis later in life.^6,7,8 In addition, each year 8% of adolescents drop out of sports activities because of injury.⁹ The reduction in physical activity resulting from sports-related injuries could have significant long-term effects on morbidity and mortality.^10,11Proprioceptive balance training is used in rehabilitation following sports-related injuries and is becoming recognized as an important element in injury prevention in sports.^{12,13,14,15,16,17,18,19} Running, jumping or pivoting on one leg relies on a sense of joint position and muscular control for joint stability. There is evidence that static balance improves following proprioceptive balance training using a wobble board.^20,21,22,23 However, most of these studies did not examine the effect of dynamic proprioceptive balance training, which may improve postural control in athletic situations and prevent some injuries.There is evidence from randomized trials that multifaceted prevention programs, including proprioceptive balance training using a wobble board, are effective in reducing injuries to the lower extremities in specific sports.^{12,13,14,15,16,17,18,19} However, the programs in these trials were multifaceted (i.e., included warm-up, flexibility, jump training, strength training, rehabilitation and sport-specific technical components), and balance was not measured. The effectiveness of balance training alone on balance ability and prevention of injury remains unclear. Moreover, the use of these techniques in adolescents and non-elite athletes has not been studied.The objectives of our study were to determine the effectiveness of a proprioceptive home-based balance-training program in improving static and dynamic balance in adolescents and to examine the effectiveness of this training program on reducing sports-related injury among adolescents.     

An approach to the management of unintentional weight loss in elderly people

UNINTENTIONAL WEIGHT LOSS, or the involuntary decline in total body weight over time, is common among elderly people who live at home. Weight loss in elderly people can have a deleterious effect on the ability to function and on quality of life and is associated with an increase in mortality over a 12-month period. A variety of physical, psychological and social conditions, along with age-related changes, can lead to weight loss, but there may be no identifiable cause in up to one-quarter of patients. We review the incidence and prevalence of weight loss in elderly patients, its impact on morbidity and mortality, the common causes of unintentional weight loss and a clinical approach to diagnosis. Screening tools to detect malnutrition are highlighted, and nonpharmacologic and pharmacologic strategies to minimize or reverse weight loss in older adults are discussed.Unintentional weight loss is the involuntary decline in total body weight over time. In clinical practice, it is encountered in up to 8% of all adult outpatients¹ and 27% of frail people 65 years and older.² Weight loss is an important risk factor in elderly patients. It is associated with increased mortality, which can range from 9% to as high as 38% within 1 to 2.5 years after weight loss has occurred.^1,3,4 Frail elderly people,⁵ people with low baseline body weight,^5,6,7 and elderly patients recently admitted to hospital are particularly susceptible to increased mortality.^8,9 Weight loss is also associated with an increased risk of in-hospital complications,^10,11 a decline in activities of daily living or physical function,^12,13 higher rates of admission to an institution^2,8 and poorer quality of life.¹⁴     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Malaria “epidemic” in Quebec: diagnosis and response to imported malaria

Background

Imported malaria is an increasing problem. The arrival of 224 African refugees presented the opportunity to investigate the diagnosis and management of imported malaria within the Quebec health care system.

Methods

The refugees were visited at home 3–4 months after arrival in Quebec. For 221, a questionnaire was completed and permission obtained for access to health records; a blood sample for malaria testing was obtained from 210.

Results

Most of the 221 refugees (161 [73%]) had had at least 1 episode of malaria while in the refugee camps. Since arrival in Canada, 87 (39%) had had symptoms compatible with malaria for which medical care was sought. Complete or partial records were obtained for 66 of these refugees and for 2 asymptomatic adults whose children were found to have malaria: malaria had been appropriately investigated in 55 (81%); no malaria smear was requested for the other 13. Smears were reported as positive for 20 but confirmed for only 15 of the 55; appropriate therapy was verified for 10 of the 15. Of the 5 patients with a false-positive diagnosis of malaria, at least 3 received unnecessary therapy. Polymerase chain reaction testing of the blood sample obtained at the home visit revealed malaria parasites in 48 of the 210 refugees (23%; 95% confidence interval [CI] 17%– 29%). The rate of parasite detection was more than twice as high among the 19 refugees whose smears were reported as negative but not sent for confirmation (47%; 95% CI 25%– 71%).

Interpretation

This study has demonstrated errors of both omission and commission in the response to refugees presenting with possible malaria. Smears were not consistently requested for patients whose presenting complaints were not “typical” of malaria, and a large proportion of smears read locally as “negative” were not sent for confirmation. Further effort is required to ensure optimal malaria diagnosis and care in such high-risk populations.In many industrialized countries, the incidence of imported malaria is rising because of changing immigration patterns and refugee policies as well as increased travel to malaria-endemic regions.^{1,2,3,4,5,6,7,8,9,10} Imported malaria is not rare in Canada (300–1000 cases per year),³ the United States^2,3,4 or other industrialized countries.^5,6,7,8,9,10 Malaria can be a serious challenge in these countries because of its potentially rapid and lethal course.^11,12,13,14 The task of front-line health care providers is made particularly difficult by the protean clinical presentations of malaria. Classic periodic fevers (tertian or quartan) are seen infrequently.^{9,15,16,17,18,19} Atypical and subtle presentations are especially common in individuals who have partial immunity (e.g., immigrants and refugees from disease-endemic areas) or are taking malaria prophylaxis (e.g., travellers).^9,16,17 Even when malaria is considered, an accurate diagnosis can remain elusive or can be delayed as a result of inadequate or distant specialized laboratory support.^19,20In Quebec, the McGill University Centre for Tropical Diseases collaborates with the Laboratoire de santé publique du Québec to raise awareness of imported malaria, to offer training and quality-assurance testing, and to provide reference diagnostic services. A preliminary diagnosis is typically made by the local laboratory, and smears (with or without staining) are sent to the McGill centre, where they are reviewed within 2–48 hours, depending on the urgency of the request. Initial medical decisions are usually based on local findings and interpretations. Although malaria is a reportable disease, there is no requirement to use the reference service.On Aug. 9, 2000, 224 refugees from Tanzanian camps landed in Montréal aboard an airplane chartered by Canadian immigration authorities. Over the ensuing 5 weeks, the McGill University Centre for Tropical Diseases noted an increase in demand for malaria reference services and an apparent small “epidemic” of imported malaria. This “epidemic” prompted us to investigate the performance of the health care system in the diagnosis and management of imported malaria.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Coinfecting Prion Strains Compete for a Limiting Cellular Resource

Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrP^Sc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrP^Sc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrP^Sc does not convert all of the available PrP^C to PrP^Sc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrP^C and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrP^Sc in animals where DY TME had completely blocked HY TME from causing disease.Prions are infectious agents of animals, including humans, which are comprised of PrP^Sc, a misfolded isoform of the noninfectious host encoded protein PrP^C (17, 24, 50, 63). Prion diseases of humans are unique neurodegenerative disorders in that they can have either a sporadic, familial, or infectious etiology. Prions cause disease in economically important domestic and wild animal species such as bovine spongiform encephalopathy in cattle and chronic wasting disease in wild and captive cervids (20, 62). Prion diseases can be zoonotic as illustrated by the transmission of bovine spongiform encephalopathy to humans that resulted in the emergence of variant Creutzfeldt-Jacob disease (14, 19, 22, 23, 46, 61, 68). Prion diseases are inevitably fatal and there are currently no effective treatments (21).Prion strains are defined by a characteristic set of features that breed true upon experimental passage (33, 34). Strain-specific differences have been identified in incubation period, clinical signs, agent distribution, overdominance, host range, neuropathology, and biochemical properties of PrP^Sc (5, 10, 11, 13, 28, 34, 42, 44). Strain-specific conformations of PrP^Sc are hypothesized to encode prion strain diversity; however, it is not understood how these differences result in the distinct strain properties (11, 19, 40, 47, 59, 66).Prion strain interference may be involved in the emergence of a dominant strain from a mixture as could occur during prion adaptation to a new host species or during prion evolution (4, 36, 43, 48, 56). In the natural prion diseases, there are examples where an individual host may be infected with more than one prion strain (15, 25, 55, 57, 58). Experimentally, coinfection or superinfection of prion strains can result in interference where a blocking, long incubation period strain extends the incubation period or completely blocks a superinfecting, short incubation period strain from causing disease (26, 27). Prion interference has been described in experimental studies of mice and hamsters infected with a wide variety of prion strains and routes of inoculation, suggesting it may be a common property of prion disease (3, 27, 52, 53, 60).It has been proposed that prion strains compete for a shared “replication site”; however, mechanistic details are not known, and it is unclear whether the blocking strain destroys or occupies the replication sites required for the superinfecting strain (28). The transport to and relative onset of replication of interfering strains in a common population of neurons is an important factor that can determine which strain will emerge (8). In the present study, we sought to determine whether the blocking strain disables transport and spread of the superinfecting strain or whether prion interference is due to competition for a cellular resource.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Cell-Cell Spread of Human Immunodeficiency Virus Type 1 Overcomes Tetherin/BST-2-Mediated Restriction in T cells

Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferon-induced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4⁺ T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.Human immunodeficiency virus type 1 can disseminate between and within hosts by cell-free infection or by direct cell-cell spread. Cell-cell spread of HIV-1 between CD4⁺ T cells is an efficient means of viral dissemination (65) and has been estimated to be several orders of magnitude more rapid than cell-free virus infection (6, 8, 41, 64, 74). Cell-cell transmission of HIV-1 takes place at the virological synapse (VS), a multimolecular structure that forms at the interface between an HIV-1-infected T cell and an uninfected target T cell during intercellular contact (27). Related structures that facilitate cell-cell spread of HIV-1 between dendritic cells and T cells (42) and between macrophages and T cells (16, 17) and for cell-cell spread of the related retrovirus human T-cell leukemia virus type 1 (HTLV-1) (24) have also been described. Moreover, more long-range cell-cell transfer can occur via cellular projections, including filopodia (71) and membrane nanotubes (75). The VS is initiated by binding of the HIV-1 envelope glycoprotein (Env), which is expressed on the surfaces of infected T cells, to HIV-1 entry receptors (CD4 and either CXCR4 or CCR5) present on the target cell membrane (6, 22, 27, 41, 61, 73). Interactions between LFA-1 and ICAM-1 and ICAM-3 further stabilize the conjugate interface and, together with Env receptor binding, help trigger the recruitment of viral proteins, CD4/coreceptor, and integrins to the contact site (27, 28, 61). The enrichment of viral and cellular proteins at the VS is an active process, dependent on cytoskeletal remodeling, and in the infected T cell both the actin and tubulin network regulate polarization of HIV-1 proteins at the cell-cell interface, thus directing HIV-1 assembly and egress toward the engaged target cell (27, 29). Virus is transferred by budding into the synaptic cleft, and virions subsequently attach to the target cell membrane to mediate entry, either by fusion at the plasma membrane or possibly following endocytic uptake (2, 22). In this way, the VS promotes more rapid infection kinetics and may enhance HIV-1 pathogenesis in vivo.Cells have evolved a number of barriers to resist invading microorganisms. One mechanism that appears to be particularly important in counteracting HIV-1 infection is a group of interferon-inducible, innate restriction factors that includes TRIM5α, APOBEC3G, and tetherin (38, 49, 69, 79). Tetherin (BST-2/CD317) is a host protein expressed by many cell types, including CD4⁺ T cells, that acts at a late stage of the HIV-1 life cycle to trap (or “tether”) mature virions at the plasma membranes of virus-producing cells, thereby inhibiting cell-free virus release (49, 56, 81). This antiviral activity of tetherin is not restricted to HIV-1, and tetherin can also inhibit the release of other enveloped viruses from infected cells (31, 40, 54, 62). What the cellular function of tetherin is besides its antiviral activity is unclear, but because expression is upregulated following alpha/beta interferon (IFN-α/β) treatment (1) and tetherin can restrict a range of enveloped viruses, tetherin has been postulated to be a broad-acting mediator of the innate immune defense against enveloped viruses.To circumvent restriction of particle release, HIV-1 encodes the 16-kDa accessory protein Vpu, which antagonizes tetherin and restores normal virus budding (47, 78). The molecular mechanisms by which Vpu does this are not entirely clear, but evidence suggests that Vpu may exert its antagonistic function by downregulating tetherin from the cell surface, trapping it in the trans-Golgi network (10) and targeting it for degradation by the proteasome (12, 39, 81) or lysosome (9, 25, 44); however, degradation of tetherin may be dispensable for Vpu activity (13), and in HIV-1-infected T cells, surface downregulation of tetherin has been reported to be minor (45), suggesting that global removal of tetherin from the plasma membrane may not be necessary to antagonize its function.Tetherin-mediated restriction of HIV-1 and antagonism by Vpu have been the focus of much research, and inhibition of cell-free virus infection has been well documented (33, 47-49, 77, 81, 82). In contrast, less studied is the impact of tetherin on direct cell-cell dissemination. For example, it is not clear if tetherin-mediated restriction inhibits T cell-T cell spread as efficiently as cell-free release or whether tetherin affects VS formation. To address these questions, we analyzed Vpu⁺ and Vpu⁻ viruses for their ability to spread directly between Jurkat T cells and primary CD4⁺ T cells in the presence or absence of endogenous tetherin. Our data suggest that tetherin does not restrict HIV-1 in the context of cell-to-cell transmission of virus between T cells expressing endogenous tetherin. Interestingly, we also that observed that Vpu-defective virus may disseminate more efficiently by cell-cell spread at the VS. We postulate that cell-cell spread may favor viral pathogenesis by allowing HIV-1 to disseminate in the presence of tetherin during an interferon-producing innate response.     

Effectiveness of statins for secondary prevention in elderly patients after acute myocardial infarction: an evaluation of class effect

Background

Clinical trials have shown the benefits of statins after acute myocardial infarction (AMI). However, it is unclear whether different statins exert a similar effect in reducing the incidence of recurrent AMI and death when used in clinical practice.

Methods

We conducted a retrospective cohort study (1997–2002) to compare 5 statins using data from medical administrative databases in 3 provinces (Quebec, Ontario and British Columbia). We included patients aged 65 years and over who were discharged alive after their first AMI-related hospital stay and who began statin treatment within 90 days after discharge. The primary end point was the combined outcome of recurrent AMI or death from any cause. The secondary end point was death from any cause. Adjusted hazard ratios (HRs) for each statin compared with atorvastatin as the reference drug were estimated using Cox proportional hazards regression analysis.

Results

A total of 18 637 patients were prescribed atorvastatin (n = 6420), pravastatin (n = 4480), simvastatin (n = 5518), lovastatin (n = 1736) or fluvastatin (n = 483). Users of different statins showed similar baseline characteristics and patterns of statin use. The adjusted HRs (and 95% confidence intervals) for the combined outcome of AMI or death showed that each statin had similar effects when compared with atorvastatin: pravastatin 1.00 (0.90–1.11), simvastatin 1.01 (0.91– 1.12), lovastatin 1.09 (0.95–1.24) and fluvastatin 1.01 (0.80– 1.27). The results did not change when death alone was the end point, nor did they change after adjustment for initial daily dose or after censoring of patients who switched or stopped the initial statin treatment.

Interpretation

Our results suggest that, under current usage, statins are equally effective for secocondary prevention in elderly patients after AMI.Randomized controlled trials (RCTs) have shown that the use of statins after acute myocardial infarction (AMI) are effective in reducing the incidence of both fatal and nonfatal cardiovascular events.^{1,2,3,4,5,6,7,8} Although these trials have significantly influenced post-AMI treatment,^9,10,11,12 it remains unclear whether all statins are equally effective in preventing recurrent AMI and death. Drugs in the same class are generally thought to be therapeutically equivalent because of similar mechanisms of action (class effect).^13,14,15 However, in the absence of comparative data, this assumption requires evaluation. Statins differ in multiple characteristics, including liver and renal metabolism, half-life, effect on other serum lipid components, bioavailability and potency.^16,17,18,19 These differences could potentially influence the extent to which the drugs are beneficial. Despite limited evidence in support of a differential benefit of statins for secondary prevention, preferential prescribing already occurs in practice and cannot be fully explained by the existing evidence or guidelines.²⁰ Comparative data of statins are thus required to inform health care decision-making.A number of RCTs have directly compared statins using surrogate end points, such as lipid reduction,^21,22,23 markers of hemostasis and inflammation^24,25,26 or reduction in number of atherotic plaques.²⁷ However, the extent to which these results can be extrapolated to clinically relevant outcomes remains to be established. The newly released PROVE IT– TIMI 22 trial²⁸ was the first trial to compare 2 statins for cardiovascular prevention. The study showed that atorvastatin used at a maximal dose of 80 mg (intensive therapy) was better than pravastatin at a dose of 40 mg (standard therapy) in decreasing the incidence of cardiovascular events and procedures. The study was, however, conducted to show the benefit associated with increased treatment intensity. It did not compare the drugs by milligram-equivalent doses or by cholesterol-lowering equivalent doses. Moreover, no difference was detected when death alone or the combined outcome of death or AMI was evaluated. Other than the PROVE IT–TIMI 22 trial, few data are currently available from RCTs that compare statins for cardiovascular prevention.²⁹We conducted a population-based study to examine the relative effectiveness of different statins for long-term secondary prevention after AMI. We used retrospective cohorts of elderly patients prescribed statins after AMI in 3 provinces. Five statins were studied: atorvastatin, pravastatin, simvastatin, lovastatin and fluvastatin. The newest statin, rosuvastatin, was not available during the study period and was not considered in this study.     

The Tumor Suppressor Functions of p27kip1 Include Control of the Mesenchymal/Amoeboid Transition

In many human cancers, p27 downregulation correlates with a worse prognosis, suggesting that p27 levels could represent an important determinant in cell transformation and cancer development. Using a mouse model system based on v-src-induced transformation, we show here that p27 absence is always linked to a more aggressive phenotype. When cultured in three-dimensional contexts, v-src-transformed p27-null fibroblasts undergo a morphological switch from an elongated to a rounded cell shape, accompanied by amoeboid-like morphology and motility. Importantly, the acquisition of the amoeboid motility is associated with a greater ability to move and colonize distant sites in vivo. The reintroduction of different p27 mutants in v-src-transformed p27-null cells demonstrates that the control of cell proliferation and motility represents two distinct functions of p27, both necessary for it to fully act as a tumor suppressor. Thus, we highlight here a new p27 function in driving cell plasticity that is associated with its C-terminal portion and does not depend on the control of cyclin-dependent kinase activity.Dissemination of tumor cells is strictly linked to their ability to attach to and move within the extracellular matrix (ECM) in a three-dimensional (3D) environment. The use of 3D experimental model systems revealed that a higher complexity in cell migration and adaptation responses exists in the 3D model than in the classical 2D model (10, 16, 41, 49). A striking example is given by the fact that only in 3D could individually migrating cells use different mechanisms such as mesenchymal and amoeboid motility (16, 17). The relative slow mesenchymal migration is characterized by a fibroblast-like spindle shape and is dependent on integrin-mediated adhesion and on protease function (16). The amoeboid motility can in some cases represent a less adhesive, integrin-independent type of movement. Cells use a propulsive mechanism and are highly deformable, and rather than degrade the matrix, they are able to squeeze through it (16). As a result, the cells that use the amoeboid motility can potentially move faster than cells that use a mesenchymal strategy. Mesenchymal and amoeboid movements are also characterized by a different involvement of small GTPases of the Rho family. A high RhoA activity is associated mainly with the amoeboid motility, while the mesenchymal migration needs a high Rac activity at the leading edge to promote the extension of cellular protrusions (41, 48). Under certain circumstances, cancer cells can undergo conversion from a mesenchymal toward an amoeboid motility, an event referred as mesenchymal-amoeboid transition (MAT) (50). MAT represents a putative escape mechanism in tumor cell dissemination that could be induced by inhibition of pericellular proteolysis (50) or by increased membrane-associated RhoA activity (18, 40).Key mediators of cell motility through ECM substrates are the members of the Src family kinases. The prototype of Src family kinases, c-Src (14), is activated following cell-ECM adhesion and contributes to regulate the focal adhesion turnover and the cytoskeletal modifications necessary for normal cell adhesion and motility (52). The c-Src gene is the proto-oncogene of the transforming gene v-src of Rous sarcoma virus, and its elevated protein level and activity have been found in many human tumors (20, 28, 27, 34). Despite the accumulation of information and new molecular understanding of how Src is controlled, there is still an incomplete picture about its role in the generation of the malignant phenotype. v-Src shows higher levels of the kinase activity and transforming ability than c-Src (14, 15, 52). It induces normal cells to acquire a variety of transformed features, including alteration of morphology and increase of invasion ability due to its role in focal adhesion remodeling (7, 9, 13).Many data suggest that there is a close relationship between cell-ECM interaction and the proliferation and movements in both normal and tumor cells (5, 38, 43). Accordingly, Src activation may influence not only cell motility but also cell cycle progression by targeting the cell cycle inhibitor p27^kip1 to proteasomal degradation (22, 39). Recent evidences indicated that p27^kip1 (hereafter called p27) can also regulate cell migration, even though its role still remains controversial since it has been reported to either block or stimulate cell movements (1, 4, 11, 19, 21, 23, 29, 45).Based on these notions, we tested the possible contribution of p27 to the growth and motility phenotypes induced by v-src transformation, with special regard to those cellular invasive features that can be observed in 3D environments. By studying in vitro and in vivo the behavior of wild-type (WT) and p27-null fibroblasts transformed with v-src, we highlight a new role for p27 in the regulation of cellular plasticity that can ultimately drive tumor cell shape, motility, and invasion.