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     

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.     

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.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

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.     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

The ESCRT-Associated Protein Alix Recruits the Ubiquitin Ligase Nedd4-1 To Facilitate HIV-1 Release through the LYPXnL L Domain Motif

The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPX_nL, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host''s fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP⁻), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP⁻ budding defects is independent of cellular Tsg101, implying that Nedd4-1''s function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPX_nL motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP⁻. This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPX_nL motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix''s facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPX_nL/Alix budding pathway via a mechanism that involves Alix ubiquitination.Retroviral Gag polyproteins bear short conserved sequences that control virus budding and release. As such, these motifs have been dubbed late or L domains (49). Three types of L domains have thus far been characterized: PT/SAP, LYPX_nL, and PPPY motifs (5, 9, 32). They recruit host proteins known to function in the vacuolar protein sorting (vps) of cargo proteins and the generation of multivesicular bodies (MVB) compartments (2). It is currently accepted that budding of vesicles into MVB involves the sequential recruitment of endosomal sorting complexes required for transport (ESCRT-I, -II, and -III) and the activity of the VPS4 AAA-ATPase (22). These sorting events are believed to be triggered by recognition of ubiquitin molecules conjugated to cargo proteins (20, 24, 41). For retrovirus budding, L domain motifs are the primary signals in Gag that elicit the recruitment of ESCRT components to facilitate viral budding. Consequently, mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase adversely affect virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (7, 10, 15, 16, 21, 28, 44).Two late domains have been identified within the p6 region of human immunodeficiency virus type 1 (HIV-1) Gag protein: the PTAP and LYPX_nL motifs. The PTAP motif binds the cellular protein Tsg101 (15, 39, 40, 47), whereas the LYPX_nL motif is the docking site for Alix (44). Tsg101 functions in HIV-1 budding (15) as a member of ESCRT-I (30, 48), a soluble complex required for the generation of MVB. This process is topologically similar to HIV-1 budding and requires the recruitment of ESCRT-III members called the charged-multivesicular body proteins (3, 29, 48) and the activity of the VPS4 AAA-ATPase (4, 48). In addition to binding the LYPX_nL motif, Alix also interacts with the nucleocapsid (NC) domain of HIV-1 Gag (13, 38), thus linking Gag to components of ESCRT-III that are critical for virus release (13).Other retroviruses, including the human T-cell leukemia virus (HTLV) and the Moloney murine leukemia virus (MoMLV), utilize the PPPY-type L domain to efficiently release virus (7, 26, 51). The PPPY motif binds members of the Nedd4-like ubiquitin ligase family (6, 7, 16, 19, 25, 43), whose normal cellular function is to ubiquitinate cargo proteins and target them into the MVB sorting pathway (11, 12, 20). Members of the Nedd4-like ubiquitin ligase family include Nedd4-1, Nedd4-2 (also known as Nedd4L), WWP-1/2, and Itch. They contain three distinct domains: an N-terminal membrane binding C2 domain (12), a central PPPY-interacting WW domain (43), and a C-terminal HECT domain that contains the ubiquitin ligase active site (42). The functional requirement for the binding of Nedd4-like ubiquitin ligases to the PPPY motif in virus budding has been demonstrated (7, 16, 18, 19, 25, 26, 28, 50, 51). Overexpression of dominant-negative mutants of Nedd4-like ligases, ESCRT-III components, or VPS4 cause a potent inhibition of PPPY-dependent virus release (7, 19, 29, 31, 52) and induce assembly and budding defects similar to those observed after perturbation of the PPPY motif (26, 51). These observations demonstrated that Nedd4-like ligases connect Gag encoding PPPY motif to ESCRT-III and VPS4 proteins to facilitate virus release.Whereas the role of Nedd4-like ubiquitin ligases in virus budding has been established, the protein interactions that link them to the cell''s ESCRT-III pathway are still unknown. Evidence for associations of Nedd4-like ligases with ESCRT proteins have been previously reported and include: the binding of Nedd4-like ubiquitin ligases LD1 and Nedd4-1 to ESCRT-I member Tsg101 (6, 31), the colocalization of multiple Nedd4-like ubiquitin ligases with endosomal compartments (1, 28), the requirement of the cell''s ESCRT pathway for Itch mediated L domain independent stimulation of MoMLV release (23), and the ubiquitination of ESCRT-I components with a shorter isoform, Nedd4-2s (8). Therefore, Nedd4-like ubiquitin ligase interactions with members of the cell''s ESCRT pathway may provide retroviral Gag with access to the host budding machinery required for virus release.Although HIV-1 Gag does not carry the PPPY canonical sequence known to interact with Nedd4-like ubiquitin ligases, both Nedd4-1 and Nedd4-2 were shown to restore the release of the HIV-1 PTAP⁻ mutant, albeit Nedd4-1 with less efficiency than Nedd4-2 (8, 46). These findings suggested that HIV-1 might utilize cellular Nedd4-like ubiquitin ligases to increase virus release. We present here evidence demonstrating that Nedd4-1 interacts with Gag and enhances HIV-1 PTAP⁻ virus release. Furthermore, we show that Nedd4-1''s function in HIV-1 release is distinct from that of Nedd4-2 in both its viral and cellular requirements. Notably, we found that Nedd4-1 enhancement of HIV-1 release requires the Alix-binding LYPX_nL L domain motif in the p6 region and basic residues in the NC domain. In addition, Alix''s facilitation of HIV-1 release requires cellular Nedd4-1, since mutations in NC that prevented Alix-mediated HIV-1 release also eliminated release by overexpression of Nedd4-1. This suggested a Nedd4-1-Alix physical and functional interdependence. In agreement with this, we found Nedd4-1 to bind and ubiquitinate Alix in the cell. Taken together, these results support a model in which Alix recruits Nedd4-1 to facilitate late steps of HIV-1 release through the LYPX_nL L domain motif via a mechanism that involves Alix ubiquitination.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).