Progressive respiratory failure is a common cause of death in patients with cystic fibrosis (CF). Although this may be related to the disease process itself, acute infectious problems may lead to respiratory failure requiring mechanical ventilation. Given the progressive nature of the disorder, some have suggested that the use of extracorporeal membrane oxygenation (ECMO) is contraindicated. The current study retrospectively reviewed the Extracorporeal Life Support Organization (ELSO) Registry to evaluate the outcomes following the use of ECMO in patients with CF. A total of 73 ECMO runs were identified in CF patients. There were 33 who received VV ECMO, 32 on VA ECMO, and 8 who received combined VA and VA ECMO. The overall survival rate for the cohort was 52% (38 of 73 patients). There was no difference in survival when comparing VA and VV ECMO. We noted an increasing trend for VV ECMO for this patient population over this time period. These data further support the need for a prospective study to evaluate outcomes following ECMO in this population with standardization of care across multiple centers.
Cystic fibrosis; extracorporeal membrane oxygenation; survival; venoarterial; venovenous
Genetic mediated physiological processes that rely on both pharmacological and nutritional principles hold great promise for the successful therapeutic targeting of reduced carbohydrate craving, body-friendly fat loss, healthy body recomposition, and overall wellness. By integrating an assembly of scientific knowledge on inheritable characteristics and environmental mediators of gene expression, we review the relationship of genes, hormones, neurotransmitters, and nutrients as they correct unwanted weight gain coupled with unhappiness. In contrast to a simple one-locus, one-mechanism focus on pharmaceuticals alone, we hypothesize that the use of nutrigenomic treatment targeting multi-physiological neurological, immunological, and metabolic pathways will enable clinicians to intercede in the process of lipogenesis by promoting lipolysis while attenuating aberrant glucose cravings. In turn, this approach will enhance wellness in a safe and predictable manner through the use of a Genetic Positioning System (GPS) Map. The GPS Map, while presently incomplete, ultimately will serve not only as a blueprint for personalized medicine in the treatment of obesity, but also for the development of strategies for reducing many harmful addictive behaviors and promoting optimal health by using substances compatible with the body’s immune system.
Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon. One of the most perplexing aspects of the C. difficile infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in C. difficile strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized Escherichia coli MazEF TA system. This E. coli MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in E. coli or Clostridium perfringens resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd in vivo, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the C. difficile transcriptome, the major C. difficile virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.
Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85 Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP). The structure shows that rPmCCAP exhibits the same haloacid dehalogenase fold and dimeric assembly as the class C enzyme from Haemophilus influenzae. Formation of the dimer in solution is demonstrated using analytical ultracentrifugation. The active site is devoid of a magnesium ion due to the presence of citrate in the crystallization buffer. Absence of the metal ion minimally perturbs the active site structure, which suggests that the main role of the ion is to balance the negative charge of the substrate rather than stabilize the active site structure. The crystal lattice displays unusual crystal packing involving the C-terminal polyhistidine tag mimicking the substrate. Steady-state kinetic constants are determined for the substrates NMN, 5´-AMP, 3´-AMP, 2´-AMP, and p-nitrophenyl phosphate. The highest catalytic efficiency is observed with NMN. The production of polyclonal anti-rPmCCAP antibodies is demonstrated, and these antibodies are shown to cross-react with the H. influenzae class C phosphatase. The antibodies are used to detect PmCCAP in clinical P. multocida and Mannheimia haemolytica strains cultured from infected animals.
X-ray crystallography; class C acid phosphatase; analytical ultracentrifugation; steady-state kinetics; polyhistidine affinity tag; haloacid dehalogenase fold
Investigation of the Vpu protein of HIV-1 recently uncovered a novel aspect of the mammalian innate response to enveloped viruses: retention of progeny virions on the surface of infected cells by the interferon-induced, transmembrane and GPI-anchored protein BST-2 (CD317; tetherin). BST-2 inhibits diverse families of enveloped viruses, but how it restricts viral release is unclear. Here, immuno-electron microscopic data indicate that BST-2 is positioned to directly retain nascent HIV virions on the plasma membrane of infected cells and is incorporated into virions. Virion-incorporation was confirmed by capture of infectivity using antibody to the ectodomain of BST-2. Consistent with a direct tethering mechanism, we confirmed that proteolysis releases restricted virions and further show that this removed the ectodomain of BST-2 from the cell surface. Unexpectedly, enzymatic cleavage of GPI anchors did not release restricted virions, weighing against models in which individual BST-2 molecules span the virion and host cell membranes. Although the exact molecular topology of restriction remains unsolved, we suggest that the incorporation of BST-2 into viral envelopes underlies its broad restrictive activity, whereas its relative exclusion from virions and sites of viral assembly by proteins such as HIV-1 Vpu may provide viral antagonism of restriction.
The cellular protein BST-2 prevents newly formed particles of HIV-1 and other enveloped viruses from escaping the infected cell by an unclear mechanism. Here, we show that BST-2 is appropriately positioned to directly retain newly formed HIV-1 virions on the cell surface and is incorporated into infectious virions. We suggest that the incorporation of BST-2 into virions is a key aspect of the protein's broadly restrictive activity against enveloped viruses.
Two members of the class C family of bacterial nonspecific acid phosphatases have been cloned, expressed, purified and crystallized. One of the crystal forms exhibited epitaxial twinning.
Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomonoesters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 Å resolution and belonged to space group C2221, with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 Å resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 Å resolution using MOSFLM and SCALA.
acid phosphatases; class C nonspecific acid phosphatases; Francisella tularensis; Pasteurella multocida; epitaxial twinning
In the current study we determined the functional significance of sodium dependent/independent glucose transporters at the neurovasculature during oxygen glucose deprivation (OGD). Confluent brain endothelial cells cocultured with astrocytes were exposed to varying degrees of in vitro stroke conditions. Glucose transporter 1 (GLUT1) and sodium glucose cotransporter (SGLT) activity were investigated by luminal membrane uptake and transport studies using [3H] D-glucose and also by [14C] alpha methyl D-glucopyranoside (AMG), a specific, nonmetabolized substrate of SGLT. In vivo middle cerebral artery occlusion (MCAO) experiments were tested to determine if blood-brain barrier (BBB) SGLT activity was induced during ischemia. Increases in luminal D-glucose and AMG uptake and transport were observed with in vitro stroke conditions. Specific inhibitor experiments suggest a combined role for both SGLT and GLUT1 at the BBB during OGD. A time dependent increase in the uptake of AMG was also seen in mice exposed to permanent focal ischemia and this increase was sensitive to SGLT inhibitor, phlorizin. Infarct and edema ratio during ischemia were significantly decreased by the inhibition of this transporter. These results show that both GLUT1 and SGLT play a role at the BBB in the blood-to-brain transport of glucose during ischemic conditions and inhibition of SGLT during stroke has the potential to improve stroke outcome. Pharmacological modulation of this novel BBB transporter could prove to be a brain vascular target in stroke.
Studies in vascular smooth muscle cells suggest that, angiotenisn II (Ang II)-mediated cellular response requires transactivation of epidermal growth factor receptor (EGF-R), and involves tyrosine phosphorylation of caveolin-1. Here we demonstrate that, exposure of WB rat liver cells to Ang II does not cause transactivation of EGF-R, but did rapidly activate p42/p44 mitogen-activated protein (MAP) kinases suggesting that it activates MAP kinases independent of EGF-R transactivation. We observed that the phospho-specific anti-caveolin-1 antibody detected a tyrosine phosphorylated, 75 kDa protein in Ang II-treated cells which we identified as glucose regulated protein-75 (GRP-75). Phosphoamino acid analysis showed that Ang II induced its phosphorylation at tyrosine, serine and threonine residues and was localized to the cytoplasm. The ability of Ang-II to induce GRP-75 phosphorylation suggests that it may play a role in the protection of cytoplasmic proteins from the damaging effect of oxidative stress known to be produced during Ang-II induced signaling.
Angiotensin II; GRP-75; Phosphorylation; Signal transduction; Oxidative stress; Mitochondria; Chaperone; EGF-R transactivation
We describe a system that permits the automated analysis of reporter gene expression in Caenorhabditis elegans with cellular resolution continuously during embryogenesis and demonstrate its utility by defining the expression patterns of reporters for several embryonically expressed transcription factors. The invariant cell lineage permits the automated alignment of multiple expression profiles, allowing the direct comparison of the expression of different genes' reporters. We have also used the system to monitor perturbations to normal development involving changes both in cell division timing and in cell fate. Systematic application could reveal the gene activity of each cell throughout development.
A review of the literature in both animals and humans reveals that changes in sex hormone have often been associated with changes in behavioral and mental abilities. Previously published research from our laboratory, and others, provides strong evidence that P300 (latency) event-related potential (ERP), a marker of neuronal processing speed, is an accurate predictor of early memory impairment in both males and females across a wide age range. It is our hypothesis, given the vast literature on the subject, that coupling growth hormones (insulin-like growth factor-I, (IGF-I) and insulin-like growth factor binding protein 3 (IGF-BP3)), P300 event-related potential and test of variables of attention (TOVA) are important neuroendocrinological predictors of early cognitive decline in a clinical setting. To support this hypothesis, we utilized structural equation modeling (SEM) parameter estimates to determine the relationship between aging and memory, as mediated by growth hormone (GH) levels (indirectly measured through the insulin-like growth factor system), P300 latency and TOVA, putative neurocognitive predictors tested in this study. An SEM was developed hypothesizing a causal directive path, leading from age to memory, mediated by IGF-1 and IGF-BP3, P300 latency (speed), and TOVA decrements. An increase in age was accompanied by a decrease in IGF-1 and IGF-BP3, an increase in P300 latency, a prolongation in TOVA response time, and a decrease in memory functioning. Moreover, independent of age, decreases in IGF-1 and IGF-BP3, were accompanied by increases in P300 latency, and were accompanied by increases in TOVA response time. Finally, increases in P300 latency were accompanied by decreased memory function, both directly and indirectly through mediation of TOVA response time. In summary, this is the first report utilizing SEM to reveal the finding that aging affects memory function negatively through mediation of decreased IGF-1 and IGF-BP3, and increased P300 latency (delayed attention and processing speed).
Structural equation modeling (SEM); P300 latency; TOVA; IGF-1; IGF-BP3; Age and memory
As socioeconomic status (SES) increases, the incidence of low birthweight and preterm birth decreases irrespective of social class. However, low birthweight remains twice as high for African-American women as for white women even when SES is controlled. This study examines to what extent second generation high SES African-American women experience improvement in birthweight and gestational age. One hundred eighty-nine former Meharry students were surveyed. Identified were 934 births that are the children and grandchildren of these students who matriculated at Meharry. These infants are compared with a cohort of white mothers from a study in the School of Public Health at Yale University. Low birthweight was reduced in the third generation high SES African-American children (6.9%) from the second generation (11.4%) but remained higher than white children (3.3%). Results showed that African-American third generation children remained at higher risk for low birthweight than were white children (relative risk [RR], 1.78; 95% confidence interval [CI], 1.03, 3.09). Similar results were observed for preterm delivery where the increased risk to third generation African-American children was 3.16 (1.89, 5.27). Persistent strong ethnic differences in birthweight in this high SES cohort (OR = 3.16, 95% CI, 1.89-5.27) support a conclusion that African-American women have birthweight distributions that are somewhat lighter than white women. This may explain a portion of current ethnic differences in birthweight. It is also possible that persistent psychosocial and behavioral factors continue to negatively influence birthweight, even in second generation high SES African-American mothers. This explanation will require identification of powerful risk factors, which are largely unrelated to those presently under investigation.
This study provides a set of nutritional and environmental parameters suitable for the growth of morel (Morchella crassipes) sclerotia in the laboratory, using a modification of the jar method of Ower et al. (U.S. patent 4,594,809, June 1986). The optimum nutritional and environmental conditions for morel sclerotium formation and maturation as determined in this study consist of a layer of rye grain supplemented with peptone, yeast extract, trace elements, and Casamino Acids overlaid with perforated aluminum foil and covered with a layer of nutrient-poor soil medium in an 8-oz. (ca. 237-ml) glass jar in the dark. We noted that addition of asparagine or aspartic acid as a nitrogen source to the rye also had a beneficial effect on sclerotium formation, while addition of carbon sources had no significant effect.
The monoclonal antibody 48G7 differs from its germline precursor by ten somatic mutations, a number of which appear to be functionally silent. We analyzed the effects of individual somatic mutations and combinations thereof on both antibody binding affinity and thermal stability. Individual somatic mutations that enhance binding affinity to hapten decrease the stability of the germline antibody; combining these binding mutations produced a mutant with high affinity for hapten but exceptionally low stability. Adding back each of the remaining somatic mutations restored thermal stability. These results, in conjunction with recently published studies, suggest an expanded role for somatic hypermutation in which both binding affinity and stability are optimized during clonal selection.
Antigenic diversity shapes immunity in distinct and unexpected ways. This is particularly true of the humoral response generated against influenza A viruses. While it is known that immunological memory developed against previously-encountered influenza A virus strains impacts the outcome of subsequent infections, exactly how sequential exposures to antigenically variant viruses shape the humoral immune response in humans remains poorly understood. To address this important question, a longitudinal analysis of antibody titers against various pandemic and seasonal strains of influenza virus spanning a 20-year period (1987–2008) was performed using samples from 40 individuals (d.o.b. 1917–1952) obtained from the Framingham Heart Study. Longitudinal increases in neutralizing antibody titers were observed against previously-encountered pandemic H2N2, H3N2 and H1N1 influenza A virus strains. Antibody titers against seasonal strains encountered later in life also increased longitudinally at a rate similar to that against their pandemic predecessors. Titers of cross-reactive antibodies specific to the hemagglutinin stalk domain were also investigated, since they are known to be influenced by exposure to antigenically diverse influenza A viruses. These titers rose modestly over time, even in the absence of major antigenic shifts. No sustained increase in neutralizing antibody titers against an antigenically more stable virus (human cytomegalovirus) was observed. The results herein describe a role for antigenic variation in shaping the humoral immune compartment, and provide a rational basis for the hierarchical nature of antibody titers against influenza A viruses in humans.
Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.
Most deaths from breast cancer result from tumor recurrence, but the mechanisms underlying tumor relapse are largely unknown. We now report that Par-4 is down-regulated during tumor recurrence and that Par-4 down-regulation is necessary and sufficient to promote recurrence. Tumor cells with low Par-4 expression survive therapy by evading a program of Par-4-dependent multinucleation and apoptosis that is otherwise engaged following treatment. Low Par-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in breast cancer patients, and Par-4 is down-regulated in residual tumor cells that survive neoadjuvant chemotherapy. Our findings identify Par-4-induced multinucleation as a mechanism of cell death in oncogene-addicted cells and establish Par-4 as a negative regulator of breast cancer recurrence.
Par-4; oncogene dependence; tumor recurrence; multinucleation
We recently identified a metabolic signature of three amino acids (tyrosine, phenylalanine, and isoleucine) that strongly predicts diabetes development. As novel modifiable targets for intervention are needed to meet the expected increase of cardiovascular disease (CVD) caused by the diabetes epidemic, we investigated whether this diabetes-predictive amino acid score (DM-AA score) predicts development of CVD and its functional consequences.
Methods and results
We performed a matched case–control study derived from the population-based Malmö Diet and Cancer Cardiovascular Cohort (MDC-CC), all free of CVD. During 12 years of follow-up, 253 individuals developed CVD and were matched for age, sex, and Framingham risk score with 253 controls. Amino acids were profiled in baseline plasma samples, using liquid chromatography-tandem mass spectrometry, and relationship to incident CVD was assessed using conditional logistic regression. We further examined whether the amino acid score also correlated with anatomical [intima-media thickness (IMT) and plaque formation] and functional (exercise-induced myocardial ischaemia) abnormalities. Compared with the lowest quartile of the DM-AA score, the odds ratio (95% confidence interval) for incident CVD in subjects belonging to quartiles 2, 3, and 4 was 1.27 (0.72–2.22), 1.96 (1.07–3.60), and 2.20 (1.12–4.31) (Ptrend = 0.010), respectively, after multivariate adjustment. Increasing quartile of the DM-AA score was cross-sectionally related to carotid IMT (Ptrend = 0.037) and with the presence of at least one plaque larger than 10 mm2 (Ptrend = 0.001). Compared with the lowest quartile of the DM-AA score, the odds ratio (95% confidence interval) for inducible ischaemia in subjects belonging to quartiles 2, 3, and 4 was 3.31 (1.05–10.4), 4.24 (1.36–13.3), and 4.86 (1.47–16.1) (Ptrend = 0.011), respectively.
This study identifies branched-chain and aromatic amino acids as novel markers of CVD development and as an early link between diabetes and CVD susceptibility.
Metabolomics; Amino acids; Diabetes; Cardiovascular disease
The phenomenon of catabolite repression enables microorganisms to use their
favourite carbon source first. New work reveals α-ketoacids as key effectors of
this process, with their levels regulating gene expression.
The HIV epidemic in Peru is concentrated primarily among men who have sex with men. HIV interventions have focused exclusively on a narrowly defined group of MSM and FSW to the exclusion of other populations potentially at increased risk. Interventions targeting MSM and FSW are insufficient and there is evidence that focusing prevention efforts solely on these populations may ignore others that do not fall directly into these categories. This paper describes non-traditional, vulnerable populations within low-income neighborhoods. These populations were identified through the use of ethnographic and epidemiologic formative research methods and the results are reported in this publication. Although the traditional vulnerable groups are still in need of prevention efforts, this study provides evidence of previously unrecognized populations at increased risk that should also receive attention from HIV/STI prevention programs.
HIV epidemiology; MSM; Sexual behavior; Risk; Community intervention trial; Vulnerability
Uncoating is an early step of HIV-1 replication in which the viral capsid disassembles by p24 capsid (p24CA) protein dissociating from the viral complex. Although uncoating is required for HIV-1 replication, many questions remain about the mechanism of this process as well as its impact on other steps in viral replication. Here we describe a recently developed assay to study the process of uncoating in HIV-1 infected cells. The CsA washout assay is a cell based assay that utilizes the HIV-1 restriction factor TRIM-CypA to detect and inhibit infection of coated viral complexes. Owl monkey kidney (OMK) cells are infected with a GFP reporter virus and TRIM-CypA restriction is switched on at various times post-infection allowing the kinetics of uncoating to be monitored in infected cells. This assay also can be used to examine the effect of different viral or cellular factors on the process of uncoating.
HIV-1; capsid; uncoating; retrovirus; CsA washout assay; TRIM-CypA
During mitotic exit missegregated chromosomes can recruit their own nuclear envelope (NE) to form micronuclei (MN). MN have reduced functioning compared to primary nuclei in the same cell, although the two compartments appear to be structurally comparable. Here we show that over 60% of MN undergo an irreversible loss of compartmentalization during interphase due to NE collapse. This disruption of the MN, which is induced by defects in nuclear lamina assembly, drastically reduces nuclear functions and can trigger massive DNA damage. MN disruption is associated with chromatin compaction and invasion of endoplasmic reticulum (ER) tubules into the chromatin. We identified disrupted MN in both major subtypes of human non-small cell lung cancer, suggesting that disrupted MN could be a useful objective biomarker for genomic instability in solid tumors. Our study shows that NE collapse is a key event underlying MN dysfunction and establishes a link between aberrant NE organization and aneuploidy.
This study examined contextual factors (caregiver depression, family resources, ethnicity, and initial levels of youth problem behavior) related to the effectiveness of the Family Check-Up (FCU) and evaluated family processes as a mediator of FCU intervention response and adolescent antisocial behavior. We followed a sample of 180 ethnically diverse youths of families who engaged in the FCU intervention. Family data were collected as part of the FCU assessment, and youth data were collected over 4 years, from sixth through ninth grade. Findings indicated that caregiver depression and minority status predicted greater caregiver motivation to change. In turn, caregiver motivation was the only direct predictor of FCU intervention response during a 1-year period. Growth in family conflict from sixth through eighth grade mediated the link between FCU response and ninth-grade antisocial behavior. This study explicitly tested core aspects of the FCU intervention model and demonstrated that caregiver motivation is a central factor that underlies family response to the FCU. The study also provided support for continued examination of family process mechanisms that account for enduring effects of the FCU and other family-centered interventions.