Infertility: A common target of antivaccine misinformation campaigns.

Misinformation, disinformation, and conspiracy theories about vaccines are key drivers of vaccine hesitancy. A repeated false claim about COVID-19 vaccines is that the vaccines cause female infertility. Dating back decades, various conspiracy theories have linked vaccination programs with infertility and thus harmed vaccination programs in Africa, Asia, and Central America, particularly against polio and tetanus. In the United States, Europe, and Australia, human papilloma virus (HPV) vaccines have been falsely blamed for infertility and primary ovarian insufficiency (POI). After distribution of COVID-19 vaccines began in December 2020, almost immediately there arose conspiracy theories claiming that these vaccines cause menstrual irregularities, miscarriages, and infertility, promoted by noted antivaccine activists Robert F. Kennedy, Jr. and Andrew Wakefield among others. Here we will explore the history of this antivaccine narrative, how it has been promulgated in the past and repurposed to COVID-19 vaccines, and strategies to counter it.

Incidental detection of malignancy on point-of-care renal ultrasound: A case series.

Renal point-of-care ultrasound (POCUS) is an increasingly common initial imaging modality in the diagnostic workup of renal colic. The primary use of renal POCUS is to assess for hydronephrosis; however, other significant findings suggestive of malignancy can also be identified. We present three cases of unexpected findings identified initially on POCUS in the emergency department, which subsequently led to new diagnoses of malignancy. As renal POCUS becomes more frequently used in clinical practice, physicians must be able to recognise abnormal images that indicate possible malignancy and the need for further workup.

Novel γ-sarcoglycan interactors in murine muscle membranes.

BACKGROUND: The sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The SC coordinates changes in phosphorylation and Ca++-flux during mechanical deformation, and these processes are disrupted with loss-of-function mutations in gamma-sarcoglycan (Sgcg) that cause Limb girdle muscular dystrophy 2C/R5. METHODS: To gain insight into how the SC mediates mechano-signaling in muscle, we utilized LC-MS/MS proteomics of SC-associated proteins in immunoprecipitates from enriched sarcolemmal fractions. Criteria for inclusion were co-immunoprecipitation with anti-Sgcg from C57BL/6 control muscle and under-representation in parallel experiments with Sgcg-null muscle and with non-specific IgG. Validation of interaction was performed in co-expression experiments in human RH30 rhabdomyosarcoma cells. RESULTS: We identified 19 candidates as direct or indirect interactors for Sgcg, including the other 3 SC proteins. Novel potential interactors included protein-phosphatase-1-catalytic-subunit-beta (Ppp1cb, PP1b) and Na+-K+-Cl--co-transporter NKCC1 (SLC12A2). NKCC1 co-localized with Sgcg after co-expression in human RH30 rhabdomyosarcoma cells, and its cytosolic domains depleted Sgcg from cell lysates upon immunoprecipitation and co-localized with Sgcg after detergent permeabilization. NKCC1 localized in proximity to the dystrophin complex at costameres in vivo. Bumetanide inhibition of NKCC1 cotransporter activity in isolated muscles reduced SC-dependent, strain-induced increases in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In silico analysis suggests that candidate SC interactors may cross-talk with survival signaling pathways, including p53, estrogen receptor, and TRIM25. CONCLUSIONS: Results support that NKCC1 is a new SC-associated signaling protein. Moreover, the identities of other candidate SC interactors suggest ways by which the SC and NKCC1, along with other Sgcg interactors such as the membrane-cytoskeleton linker archvillin, may regulate kinase- and Ca++-mediated survival signaling in skeletal muscle.

Oral administration of a synthetic vinyl-ether plasmalogen normalizes open field activity in a mouse model of rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare genetic disorder caused by mutations in peroxisomal genes essential for plasmalogen biosynthesis. Plasmalogens are a class of membrane glycerophospholipids containing a vinyl-ether-linked fatty alcohol at the sn-1 position that affect functions including vesicular transport, membrane protein function and free radical scavenging. A logical rationale for the treatment of RCDP is therefore the therapeutic augmentation of plasmalogens. The objective of this work was to provide a preliminary characterization of a novel vinyl-ether synthetic plasmalogen, PPI-1040, in support of its potential utility as an oral therapeutic option for RCDP. First, wild-type mice were treated with 13C6-labeled PPI-1040, which showed that the sn-1 vinyl-ether and the sn-3 phosphoethanolamine groups remained intact during digestion and absorption. Next, a 4-week treatment of adult plasmalogen-deficient Pex7hypo/null mice with PPI-1040 showed normalization of plasmalogen levels in plasma, and variable increases in plasmalogen levels in erythrocytes and peripheral tissues (liver, small intestine, skeletal muscle and heart). Augmentation was not observed in brain, lung and kidney. Functionally, PPI-1040 treatment normalized the hyperactive behavior observed in the Pex7hypo/null mice as determined by open field test, with a significant inverse correlation between activity and plasma plasmalogen levels. Parallel treatment with an equal amount of ether plasmalogen precursor, PPI-1011, did not effectively augment plasmalogen levels or reduce hyperactivity. Our findings show, for the first time, that a synthetic vinyl-ether plasmalogen is orally bioavailable and can improve plasmalogen levels in an RCDP mouse model. Further exploration of its clinical utility is warranted.This article has an associated First Person interview with the joint first authors of the paper.

Neuroprotection and immunomodulation in the gut of parkinsonian mice with a plasmalogen precursor.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. It is typically associated with motor symptoms originating from the degeneration of nigrostriatal dopamine (DA) neurons. Early stages of PD have been associated with an alteration in DA production in intestinal DAergic neurons along with inflammation. Interestingly, decreased serum concentrations of ethanolamine plasmalogens (PlsEtn) have been reported in PD patients. Ethanolamine plasmalogens play a role in vesicular fusion and release during neurotransmission, and store neuroprotective polyunsaturated fatty acids, such as docosahexaenoic acid (DHA) and are strong anti-oxidants, highlighting areas of potential therapeutic interest. Docosahexaenoic acid is known to play important roles in both the central nervous and peripheral systems, in addition to acting as a precursor of several molecules that regulate the resolution of inflammation. The present study investigated the neuroprotective and anti-inflammatory properties of the DHA-containing PlsEtn precursor, PPI-1011, in the intestine of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Treatment with PPI-1011 prevented the MPTP-induced decrease in PlsEtn levels. In addition it prevented the loss of tyrosine hydroxylase (TH) expression and reduced the infiltration of macrophages in the myenteric plexus of MPTP-treated mice. The protective effects of PPI-1011 were observed regardless of whether it was administered pre- or post- MPTP treatment. These results suggest that PPI-1011 has neuroprotective and anti-inflammatory properties in the gut and indicate its potential utility as a treatment for both early and more advanced stages of PD.

Performance evaluation of the Bio-Rad Geenius HIV 1/2 supplemental assay.

BACKGROUND: In the US, the HIV diagnostic algorithm for laboratory settings recommends the use of an HIV-1/HIV-2 differentiation supplemental assay after an initial reactive antigen/antibody (Ag/Ab) assay result. Since the discontinuation of the Multispot HIV-1/HIV-2 Rapid Test (MS), the Geenius HIV-1/2 Supplemental assay (Geenius) is the only FDA-approved supplemental differentiation test. OBJECTIVE: We compared the performance of Geenius to MS and Western Blot (WB). STUDY DESIGN: The relative seroconversion plasma reactivity of Geenius and MS was assessed using a 50% cumulative frequency analysis from 17 HIV-1 seroconverters. In addition, previously characterized plasma specimens, 186 HIV-1 positive, 100 HIV-2 positive, and 93 Ag/Ab-positive/HIV-1 RNA-negative, were tested with Geenius v1.1 software. McNemar's test was used for paired comparison analysis. A subset of 48 specimens were retested with the upgraded Geenius v1.3 software. RESULTS: In HIV-1 seroconverters, the relative seroconversion reactivity was 2.5 and 2 days before the first positive HIV-1 WB for Geenius and MS, respectively. In HIV-1 positive samples, Geenius performed similarly to HIV-1 WB (p=0.1687) and MS (p=0.8312). In HIV-2 positive samples, Geenius underperformed compared to HIV-2 WB (p=0.0005) and MS (p=0.0012). When using the upgraded software among the HIV-1 positive and Ag/Ab-reactive/HIV-1 RNA-negative samples, gp140 reactivity decreased without affecting characterization of HIV-2 samples. CONCLUSIONS: With HIV-1 samples, Geenius, WB and MS performance was similar as supplemental tests. The updated Geenius software reduced false gp140 reactivity, but had no impact on identifying true HIV-2 infections. Further evaluation will assess the impact of the Geenius software update on final diagnostic interpretations.

Generation and characterization of monoclonal antibodies that recognize human and murine supervillin protein isoforms.

Supervillin isoforms have been implicated in cell proliferation, actin filament-based motile processes, vesicle trafficking, and signal transduction. However, an understanding of the roles of these proteins in cancer metastasis and physiological processes has been limited by the difficulty of obtaining specific antibodies against these highly conserved membrane-associated proteins. To facilitate research into the biological functions of supervillin, monoclonal antibodies were generated against the bacterially expressed human supervillin N-terminus. Two chimeric monoclonal antibodies with rabbit Fc domains (clones 1E2/CPTC-SVIL-1; 4A8/CPTC-SVIL-2) and two mouse monoclonal antibodies (clones 5A8/CPTC-SVIL-3; 5G3/CPTC-SVIL-4) were characterized with respect to their binding sites, affinities, and for efficacy in immunoblotting, immunoprecipitation, immunofluorescence microscopy and immunohistochemical staining. Two antibodies (1E2, 5G3) recognize a sequence found only in primate supervillins, whereas the other two antibodies (4A8, 5A8) are specific for a more broadly conserved conformational epitope(s). All antibodies function in immunoblotting, immunoprecipitation and in immunofluorescence microscopy under the fixation conditions identified here. We also show that the 5A8 antibody works on immunohistological sections. These antibodies should provide useful tools for the study of mammalian supervillins.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation.

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer.

Resistance to zinc and cadmium in Staphylococcus aureus of human and animal origin.

OBJECTIVE: Studies conducted in Europe have observed resistance to trace metals such as zinc chloride and copper sulfate in livestock-associated Staphylococcus aureus. This study was conducted to determine the prevalence of zinc and cadmium resistance in S. aureus isolated in the United States. DESIGN: Cross-sectional study of convenience sample of S. aureus isolates. PARTICIPANTS: Three hundred forty-nine S. aureus isolates, including methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) obtained from human, swine, and retail meat were included in the sample set. METHODS: Polymerase chain reaction was used to test for the presence of genes for zinc and cadmium resistance (czrC), methicillin resistance (mecA), and staphylococcal complement inhibitor (scn). Antibiotic susceptibility of isolates was tested using the broth microdilution method. Data were analyzed using the multivariable logistic regression method. RESULTS: Twenty-nine percent (102/349) of S. aureus isolates were czrC positive. MRSA isolates were more likely to be czrC positive compared to MSSA (MRSA czrC positive: 12/61, 19.6%; MSSA czrC positive: 12/183, 6.6%). After adjustment for oxacillin and clindamycin susceptibility in analysis, multidrug-resistant S. aureus was observed to have low odds of being czrC positive (P = .03). The odds of being czrC positive were observed to be significantly high in tetracycline-resistant S. aureus isolated from noninfection samples (P = .009) and swine (P < .0001). CONCLUSIONS: Resistance to zinc and cadmium was observed to be associated with MRSA, a finding consistently observed in European studies. Prolonged exposure to zinc in livestock feeds and fertilizers could propagate resistance to the metal ion, thereby hindering use of zinc-based topical agents in treating S. aureus infections.

Human genome-wide association and mouse knockout approaches identify platelet supervillin as an inhibitor of thrombus formation under shear stress.

BACKGROUND: High shear force critically regulates platelet adhesion and thrombus formation during ischemic vascular events. To identify genetic factors that influence platelet thrombus formation under high shear stress, we performed a genome-wide association study and confirmatory experiments in human and animal platelets. METHODS AND RESULTS: Closure times in the shear-dependent platelet function analyzer (PFA)-100 were measured on healthy, nondiabetic European Americans (n=125) and blacks (n=116). A genome-wide association (P<5×10(-8)) was identified with 2 single-nucleotide polymorphisms within the SVIL gene (chromosome 10p11.23) in African Americans but not European Americans. Microarray analyses of human platelet RNA demonstrated the presence of SVIL isoform 1 (supervillin) but not muscle-specific isoforms 2 and 3 (archvillin, SmAV). SVIL mRNA levels were associated with SVIL genotypes (P≤0.02) and were inversely correlated with PFA-100 closure times (P<0.04) and platelet volume (P<0.02). Leukocyte-depleted platelets contained abundant levels of the ≈205-kDa supervillin polypeptide. To assess functionality, mice lacking platelet supervillin were generated and back-crossed onto a C57BL/6 background. Compared with controls, murine platelets lacking supervillin were larger by flow cytometry and confocal microscopy and exhibited enhanced platelet thrombus formation under high-shear but not low-shear conditions. CONCLUSIONS: We show for the first time that (1) platelets contain supervillin; (2) platelet thrombus formation in the PFA-100 is associated with human SVIL variants and low SVIL expression; and (3) murine platelets lacking supervillin exhibit enhanced platelet thrombus formation at high shear stress. These data are consistent with an inhibitory role for supervillin in platelet adhesion and arterial thrombosis.

High prevalence of Panton-Valentine leukocidin among methicillin-sensitive Staphylococcus aureus colonization isolates in rural Iowa.

Recent studies have shown that livestock can carry Staphylococcus aureus and transmit it to human caretakers. We conducted a pilot study to determine the prevalence and molecular epidemiology of S. aureus among rural Iowans, including individuals with livestock contact. Nasal and throat swabs were collected and plated onto selective media to isolate methicillin-susceptible and methicillin-resistant S. aureus (MRSA), followed by antibiotic resistance testing and molecular analysis of the isolates. While no MRSA was detected, overall, 23.7% (31/131) of participants were found to harbor S. aureus in their nose, throat, or both. Fifteen isolates displayed resistance to one or more tested antibiotics, and the Panton-Valentine leukocidin (PVL) genes were present at a high level (29% [9/31] of S. aureus-positive participants). Younger age and tobacco use were associated with increased risk of S. aureus carriage. Our results suggest that carriage of PVL-positive S. aureus is common among rural Iowans, even in the absence of detectable MRSA colonization.

Oral bioavailability of the ether lipid plasmalogen precursor, PPI-1011, in the rabbit: a new therapeutic strategy for Alzheimer's disease.

INTRODUCTION: Docosahexaenoic acid (DHA) and DHA-containing ethanolamine plasmalogens (PlsEtn) are decreased in the brain, liver and the circulation in Alzheimer's disease. Decreased supply of plasmalogen precursors to the brain by the liver, as a result of peroxisomal deficits is a process that probably starts early in the AD disease process. To overcome this metabolic compromise, we have designed an orally bioavailable DHA-containing ether lipid precursor of plasmalogens. PPI-1011 is an alkyl-diacyl plasmalogen precursor with palmitic acid at sn-1, DHA at sn-2 and lipoic acid at sn-3. This study outlines the oral pharmacokinetics of this precursor and its conversion to PlsEtn and phosphatidylethanolamines (PtdEtn). METHODS: Rabbits were dosed orally with PPI-1011 in hard gelatin capsules for time-course and dose response studies. Incorporation into PlsEtn and PtdEtn was monitored by LC-MS/MS. Metabolism of released lipoic acid was monitored by GC-MS. To monitor the metabolic fate of different components of PPI-1011, we labeled the sn-1 palmitic acid, sn-2 DHA and glycerol backbone with (13)C and monitored their metabolic fates by LC-MS/MS. RESULTS: PPI-1011 was not detected in plasma suggesting rapid release of sn-3 lipoic acid via gut lipases. This conclusion was supported by peak levels of lipoic acid metabolites in the plasma 3 hours after dosing. While PPI-1011 did not gain access to the plasma, it increased circulating levels of DHA-containing PlsEtn and PtdEtn. Labeling experiments demonstrated that the PtdEtn increases resulted from increased availability of DHA released via remodeling at sn-2 of phospholipids derived from PPI-1011. This release of DHA peaked at 6 hrs while increases in phospholipids peaked at 12 hr. Increases in circulating PlsEtn were more complex. Labeling experiments demonstrated that increases in the target PlsEtn, 16:0/22:6, consisted of 2 pools. In one pool, the intact precursor received a sn-3 phosphoethanolamine group and desaturation at sn-1 to generate the target plasmalogen. The second pool, like the PtdEtn, resulted from increased availability of DHA released during remodeling of sn-2. In the case of sn-1 18:0 and 18:1 plasmalogens with [(13)C(3)]DHA at sn-2, labeling was the result of increased availability of [(13)C(3)]DHA from lipid remodeling. Isotope and repeated dosing (2 weeks) experiments also demonstrated that plasmalogens and/or plasmalogen precursors derived from PPI-1011 are able to cross both the blood-retinal and blood-brain barriers. CONCLUSIONS: Our data demonstrate that PPI-1011, an ether lipid precursor of plasmalogens is orally bioavailable in the rabbit, augmenting the circulating levels of unesterified DHA and DHA-containing PlsEtn and PtdEtn. Other ethanolamine plasmalogens were generated from the precursor via lipid remodeling (de-acylation/re-acylation reactions at sn-2) and phosphatidylethanolamines were generated via de-alkylation/re-acylation reactions at sn-1. Repeated oral dosing for 2 weeks with PPI-1011 resulted in dose-dependent increases in circulating DHA and DHA-containing plasmalogens. These products and/or precursors were also able to cross the blood-retinal and blood-brain barriers.

Cellular diamine levels in cancer chemoprevention: modulation by ibuprofen and membrane plasmalogens.

BACKGROUND: To develop effective strategies in cancer chemoprevention, an increased understanding of endogenous biochemical mediators that block metastatic processes is critically needed. Dietary lipids and non-steroidal anti-inflammatory drugs (NSAIDs) have a published track record of providing protection against gastrointestinal malignancies. In this regard, we examined the effects of membrane plasmalogens and ibuprofen on regulation of cellular levels of diamines, polyamine mediators that are augmented in cancer cells. For these studies we utilized Chinese hamster ovary (CHO) cells and NRel-4 cells, a CHO cell line with defective plasmalogen synthesis. RESULTS: NRel-4 cells, which possess cellular plasmalogen levels that are 10% of control CHO cells, demonstrated 2- to 3-fold increases in cellular diamine levels. These diamine levels were normalized by plasmalogen replacement and significantly reduced by ibuprofen. In both cases the mechanism of action appears to mainly involve increased diamine efflux via the diamine exporter. The actions of ibuprofen were not stereospecific, supporting previous studies that cyclooxygenase (COX) inhibition is unlikely to be involved in the ability of NSAIDs to reduce intracellular diamine levels. CONCLUSIONS: Our data demonstrate that ibuprofen, a drug known to reduce the risk of colorectal cancer, reduces cellular diamine levels via augmentation of diamine efflux. Similarly, augmentation of membrane plasmalogens can increase diamine export from control and plasmalogen-deficient cells. These data support the concept that membrane transporter function may be a therapeutic point of intervention for dietary and pharmacological approaches to cancer chemoprevention.

In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor.

BACKGROUND: Childhood peroxisomal disorders and leukodystrophies are devastating diseases characterized by dysfunctional lipid metabolism. Plasmalogens (ether glycerophosphoethanolamine lipids) are decreased in these genetic disorders. The biosynthesis of plasmalogens is initiated in peroxisomes but completed in the endoplasmic reticulum. We therefore undertook a study to evaluate the ability of a 3-substituted, 1-alkyl, 2-acyl glyceryl ether lipid (PPI-1011) to replace plasmalogens in rhizomelic chrondrodysplasia punctata type 1 (RCDP1) and rhizomelic chrondrodysplasia punctata type 2 (RCDP2) lymphocytes which possess peroxisomal mutations culminating in deficient plasmalogen synthesis. We also examined plasmalogen synthesis in Pelizaeus-Merzbacher disease (PMD) lymphocytes which possess a proteolipid protein-1 (PLP1) missense mutation that results in abnormal PLP1 folding and it's accumulation in the endoplasmic reticulum (ER), the cellular site of the last steps in plasmalogen synthesis. In vivo incorporation of plasmalogen precursor into tissue plasmalogens was also evaluated in the Pex7 mouse model of plasmalogen deficiency. RESULTS: In both RCDP1 and RCDP2 lymphocytes, PPI-1011 repleted the target ethanolamine plasmalogen (PlsEtn16:0/22:6) in a concentration dependent manner. In addition, deacylation/reacylation reactions resulted in repletion of PlsEtn 16:0/20:4 in both RCDP1 and RCDP2 lymphocytes, repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP2 lymphocytes, and partial repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP1 lymphocytes. In the Pex7 mouse, oral dosing of labeled PPI-1011 demonstrated repletion of tissue levels of the target plasmalogen PlsEtn 16:0/22:6 with phospholipid remodeling also resulting in significant repletion of PlsEtn 16:0/20:4 and PlsEtn 16:0/18:1. Metabolic conversion of PPI-1011 to the target plasmalogen was most active in the liver. CONCLUSIONS: Our data demonstrate that PPI-1011 is activated (removal of 3-substitution) and converted to PlsEtn in vitro in both RCDP1 and RCDP2 lymphocytes and in vivo in the Pex7 mouse model of RCPD1 effectively bypassing the peroxisomal dysfunction present in these disorders. While PPI-1011 was shown to replete PlsEtns 16:0/x, ether lipid precursors of PlsEtn 18:0/x and PlsEtn 18:1/x may also be needed to achieve optimal clinical benefits of plasmalogen replacement in these complex patient populations. In contrast, only limited plasmalogen replacement was observed in PMD lymphocytes suggesting that the effects of protein misfolding and accumulation in the ER negatively affect processing of plasmalogen precursors in this cellular compartment.

Detection of N-acetyl methionine in human and murine brain and neuronal and glial derived cell lines.

Despite the fact that N-acetyl methionine (NAM) supplementation has long been reported as a bioavailable source of methionine in humans, and known to reduce liver toxicity after acetaminophen overdose, its cellular endogenous presence has never been investigated. We demonstrate for the first time that NAM is present in both human and mouse tissues and cells in culture. A wide variety of cultured cells, including a number of brain derived cell types, as well as mouse and human brain tissue all have clearly detectable levels of NAM. Methionine is rapidly acetylated to form NAM in cultured human oligodendroglioma cells with an initial rate of 0.44 ± 0.064 atom percent excess per minute. The presence of measurable quantities of NAM in brain cells in combination with its rapid formation point to a potential physiological role for N-acetylated methionine in the brain. Aminoacylase 1 is responsible for metabolism of NAM to methionine and acetate. Deficiencies in aminoacylase 1 have been linked to a variety of neurological disorders; however, it is unclear whether and how the brain is affected by this defect. The reported presence of NAM in the human brain may provide an invaluable key to discovering the link between aminoacylase 1 mutations and neurological problems.

Novel interactors and a role for supervillin in early cytokinesis.

Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2-G3 or G4-G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle.

The membrane-associated protein, supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility.

In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F-actin- and myosin II-binding protein that tightly associates with signaling proteins in cholesterol-rich, 'lipid raft' membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin-knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed-end inhibitor, cytochalasin D, suggests that both treatments affect actin-dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal-regulated kinases (ERKs) 1 and 2 and increases the velocity of cell translocation. These results suggest that supervillin, F-actin and associated proteins coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin-based cell motility.

Supervillin reorganizes the actin cytoskeleton and increases invadopodial efficiency.

Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface.

Smoking in film in New Zealand: measuring risk exposure.

BACKGROUND: Smoking in film is a risk factor for smoking uptake in adolescence. This study aimed to quantify exposure to smoking in film received by New Zealand audiences, and evaluate potential interventions to reduce the quantity and impact of this exposure. METHODS: The ten highest-grossing films in New Zealand for 2003 were each analysed independently by two viewers for smoking, smoking references and related imagery. Potential interventions were explored by reviewing relevant New Zealand legislation, and scientific literature. RESULTS: Seven of the ten films contained at least one tobacco reference, similar to larger film samples. The majority of the 38 tobacco references involved characters smoking, most of whom were male. Smoking was associated with positive character traits, notably rebellion (which may appeal to adolescents). There appeared to be a low threshold for including smoking in film. Legislative or censorship approaches to smoking in film are currently unlikely to succeed. Anti-smoking advertising before films has promise, but experimental research is required to demonstrate cost effectiveness. CONCLUSION: Smoking in film warrants concern from public health advocates. In New Zealand, pre-film anti-smoking advertising appears to be the most promising immediate policy response.

Supervillin modulation of focal adhesions involving TRIP6/ZRP-1.

Cell-substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)--a peripheral membrane protein that binds myosin II and F-actin in such cells--negatively regulates stress fibers, FAs, and cell-substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor-interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV-TRIP6 interaction may regulate FA maturation and/or disassembly.