Premature Ventricular Contractions and Non-sustained Ventricular Tachycardia: Association with Sudden Cardiac Death,Risk Stratification,and Management Strategies

Premature ventricular contractions (PVCs) and non-sustained ventricular tachycardia (NSVT) are frequently encountered and a marker of electrocardiomyopathy. In some instances, they increase the risk for sustained ventricular tachycardia, ventricular fibrillation, and sudden cardiac death. While often associated with a primary cardiomyopathy, they have also been known to cause tachycardia-induced cardiomyopathy in patients without preceding structural heart disease. Medical therapy including beta-blockers and class III anti-arrhythmic agents can be effective while implantable cardiac defibrillators (ICD) are indicated in certain patients. Radiofrequency ablation (RFA) is the preferred, definitive treatment in those patients that improve with anti-arrhythmic therapy, have tachycardia-induced cardiomyopathy, or have certain subtypes of PVCs/NSVT. We present a review of PVCs and NSVT coupled with case presentations on RFA of fascicular ventricular tachycardia, left-ventricular outflow tract ventricular tachycardia, and Purkinje arrhythmia leading to polymorphic ventricular tachycardia.     

Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.     

Clonal Expansion of Normal-Appearing Human Hepatocytes during Chronic Hepatitis B Virus Infection

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.Transient hepatitis B virus (HBV) infections, which generally last <6 months, do not cause cirrhosis and cause only minor increases in the risk of hepatocellular carcinoma (HCC) (3, 46). Chronic infections, typically lifelong, can cause cirrhosis and HCC (3). Of the ∼350 million HBV carriers now alive, ca. 60 million will die prematurely of cirrhosis and/or HCC. Cirrhosis, which usually develops late in infection, is a significant risk factor for HCC. Early reports stated that most HCCs occur on a background of cirrhosis. However, later studies suggested that as many as 50% of HCCs may occur in noncirrhotic liver (4), that is, in patients in whom the progression of liver disease still appears rather mild. Thus, liver damage that appears severe by histologic examination is not a prerequisite for HCC.Interestingly, during chronic HBV infections there is, in the face of persistent viremia, a decline over time in the fraction of infected hepatocytes, from 100% to as little as a few percent (5, 12-14, 16, 17, 22, 23, 27, 34, 37, 38). Along with HCC, this is perhaps the most surprising and unexplained outcome of chronic infection. The timing of this decline has not been systematically studied, but it is presumably gradual, occurring over years or decades, and dependent on persistent, albeit low-level, killing of infected hepatocytes by antiviral cytotoxic T lymphocytes (CTLs) (20). It is believed that the liver is largely a closed, self-renewing population. Such a population might be expected to evolve under any strong or persistent selective pressure. In HBV-infected patients, the earliest and most persistent selective pressure is immune killing of infected hepatocytes, which should initially constitute the entire hepatocyte population. Persistent killing of HBV-infected hepatocytes could lead to clonal expansion of mutant or epigenetically altered hepatocytes that had lost the ability to support infection and that were not, therefore, targeted by antiviral CTLs.Such a selective pressure may explain why foci of altered hepatocytes (FAH) and HCC are typically virus negative (1, 6, 11, 26, 29, 31, 35, 40, 41, 44). Normal or preneoplastic hepatocytes (e.g., in FAH) that have evaded the host immune response should undergo clonal expansion, because their death rate is lower than that of surrounding hepatocytes, even if they do not have a higher growth rate. Indeed, clonal expansion of hepatocytes has been detected, in the absence of cirrhosis, in woodchucks chronically infected with woodchuck hepatitis virus (WHV) (19) and in chimpanzees chronically infected with HBV (21). The presence of discrete foci of normal-appearing but virus-negative hepatocytes in chronically infected woodchuck livers (39) suggested, but did not prove, that normal-appearing hepatocytes that had lost the ability to support virus replication might clonally expand.The purpose of the present study was, therefore, to determine if normal-appearing hepatocytes undergo clonal expansion. To address this issue, we focused on noncirrhotic livers, because hepatocyte appearance and organization in many cirrhotic nodules are often considered to indicate premalignancy (7, 24, 25, 44), and this, together with the cellular environment in the cirrhotic liver, may explain why as many as 50% of cirrhotic nodules have been found to be made up of clonally expanded hepatocytes (2, 18, 24, 25, 28, 44). In older HBV patients, cirrhosis, the result of cumulative scarring due to ongoing tissue injury, presumably produces an evolutionary pressure on the hepatocyte population due to restricted blood flow and altered hepatic architecture.Clonal expansion was detected by assaying for integrated HBV DNA by inverse PCR (19, 21). Because integration occurs at random sites in host DNA, each integration event provides a unique genetic marker for the cell in which it occurred, and for any daughter cells. Thus, the clonal expansion of these tagged hepatocytes can be measured by determining how many times a given virus-cell DNA junction is repeated in a liver fragment. Analysis of fragments of nontumorous liver from noncirrhotic HCC patients revealed that at least 1% of hepatocytes are present as clones of >1,000 cells. Examination of 5-μm-thick sections of paraffin-embedded livers from the same patients revealed that clonally expanded hepatocytes were present in liver sections lacking preneoplastic lesions or changes. Therefore, normal-appearing hepatocytes must have undergone clonal expansion. Although clonal expansion was detected by analysis of integrated HBV DNA, the expansion did not appear to be due to the site of integration of the viral DNA into host DNA.These results are consistent with the hypothesis that immune selection and the later emergence of liver cirrhosis, with altered lobular organization and restricted blood flow, may constitute the two major selective pressures on the hepatocyte population that culminate in hepatocellular carcinoma. More-direct proof of the role, if any, of immune selection in hepatocyte evolution and HCC will require, first of all, an assay with a greater ability to detect clonally expanded hepatocytes. The present approach is limited by a number of factors, including a need for integration near a particular restriction endonuclease cleavage site in host DNA and for conservation of particular viral sequences so that the integrated DNA can be amplified using the PCR primers chosen. These issues may explain why the fraction of clonally expanded hepatocytes reported here is much less than that suggested by histologic data showing that more than 50% of hepatocytes appear negative for virus replication in long-term carriers. Further dissection of this issue will also require localization and determination of the virologic status of hepatocyte clones present in tissue sections.     

Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid ��-Spectrin in a Calcium- and Calmodulin-dependent Manner

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph^1J/sph^1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G₃ peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K_d = 0.30 μm). EF-domains bearing the sph^1J mutation are inactive. Binding of protein 4.2 to band 3 (K_d = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca²⁺ (but not Mg²⁺) by three to five times. Calmodulin also binds to the EF-domain (K_d = 17 μm), and Ca²⁺-calmodulin blocks Ca²⁺-dependent binding of protein 4.2 but not Ca²⁺-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.     

Human plasma membrane-derived vesicles halt proliferation and induce differentiation of THP-1 acute monocytic leukemia cells

Plasma membrane-derived vesicles (PMVs) are small intact vesicles released from the cell surface that play a role in intercellular communication. We have examined the role of PMVs in the terminal differentiation of monocytes. The myeloid-differentiating agents all-trans retinoic acid/PMA and histamine, the inflammatory mediator that inhibits promonocyte proliferation, induced an intracellular Ca(2+)-mediated PMV (as opposed to exosome) release from THP-1 promonocytes. These PMVs cause THP-1 cells to enter G(0)-G(1) cell cycle arrest and induce terminal monocyte-to-macrophage differentiation. Use of the TGF-β receptor antagonist SB-431542 and anti-TGF-β1 Ab showed that this was due to TGF-β1 carried on PMVs. Although TGF-β1 levels have been shown to increase in cell culture supernatants during macrophage differentiation and dendritic cell maturation, the presence of TGF-β1 in PMVs is yet to be reported. In this study, to our knowledge we show for the first time that TGF-β1 is carried on the surface of PMVs, and we confirm the presence within PMVs of certain leaderless proteins, with reported roles in myeloid cell differentiation. Our in vitro findings support a model in which TGF-β1-bearing PMVs, released from promonocytic leukemia cells (THP-1) or primary peripheral blood monocytes on exposure to sublytic complement or after treatment with a differentiation therapy agent, such as all-trans retinoic acid, significantly reduce proliferation of THP-1 cells. Such PMVs also induce the terminal differentiation of primary peripheral blood monocytes as well as THP-1 monocytes.     

The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis

The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.Shewanella oneidensis strain MR-1 (MR-1) is a facultative anaerobe capable of respiring a variety of substrates, including various metals and metal oxides, a phenotype that is important for bioremediation and metal cycling in natural environments (22, 53). At near-neutral pH, Fe(III) and Mn(IV) often are present as insoluble oxide minerals. Dissimilatory metal-reducing bacteria such as MR-1 have developed pathways to transfer electrons from the interior of the cell to these external terminal electron acceptors. In some bacteria, these pathways also can transfer electrons to electrodes, which can be harnessed for renewable energy and remote biosensor applications (23, 26, 27). Beyond increasing our understanding of this unusual process, applying anaerobic microbial extracellular respiration to new technologies requires a thorough understanding of the molecular dynamics and cellular physiology of electron source utilization (substrate oxidation) and the reduction of insoluble terminal electron acceptor(s). There are four proposed mechanisms to explain how insoluble substrates are reduced by Shewanella: (i) direct contact, (ii) electron shuttling, (iii) chelation, and (iv) electrically conductive appendages (reviewed in reference 18). We will focus on the first three strategies here.Flavins recently have been discovered to accelerate the reduction of both iron oxide minerals (51) and electrodes (30) by MR-1. Riboflavin (vitamin B₂) is a precursor for the biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (13). Riboflavin and FMN both can be observed to build up in the supernatant of anaerobic and aerobically grown cultures of MR-1 (30, 51). However, the mechanism by which flavins enhance the rate of iron oxide mineral or electrode reduction is unknown, although recent work is consistent with a critical role for these compounds in mediating solid Fe(III) reduction by MR-1 (42). Since soluble (chelated) Fe(III) is reduced faster than insoluble Fe(III) by MR-1 (6), one possible explanation for the enhancement of insoluble iron reduction by flavins is increased available soluble iron via chelation (1, 2, 30). Flavins also may be utilized as redox-active compounds to traffic electrons between extracellular reductases on the surface of the cell and insoluble substrates (30, 51), a process termed electron shuttling (18, 39, 41). The chelation of the terminal electron acceptor during electrode reduction is not relevant when the anode is composed of graphite. Therefore, electron shuttling likely is responsible for the flavin enhancement of current production on poised-potential electrodes (30). However, it is unclear if the chelation of metals by flavins influences insoluble metal reduction by S. oneidensis (30).The Mtr pathway is required for the reduction of metals and electrodes (5, 6, 9, 17). Five primary protein components have been identified in this pathway: OmcA, MtrC, MtrA, MtrB, and CymA (47). Current models of electron transfer in MR-1 assume that electrons from carbon source oxidation are passed via the menaquinone pool to the inner membrane-anchored c-type cytochrome CymA (19, 31). These electrons then are transferred to a periplasmic c-type cytochrome, MtrA, and eventually to outer membrane (OM)-anchored c-type cytochromes MtrC and OmcA, which interact with an integral OM scaffolding protein, MtrB (32, 33, 43). These OM cytochromes then can reduce various substrates, including iron oxides and electrodes (8, 9, 12, 36, 47). Since the Mtr system is required by MR-1 to reduce many different substrates (18), it also could be capable of reducing extracellular flavins. Indeed, electron transfer to carbon electrodes is impaired in strains lacking Mtr pathway components (9, 17), which may be explained simply by a decreased ability to reduce extracellular flavins. The observation that Mtr mutants produce less current on electrodes than the wild type could be due to (i) less current generated per cell (either direct reduction or flavin mediated), (ii) decreased attachment to the electrode surface, (iii) differences in external flavin concentrations, or (iv) a combination of these three possibilities. Determining the specific activity (current produced per unit of attached biomass) of Mtr mutants on electrodes under conditions where flavin levels were controlled would allow for differentiation between these possibilities. To date, this kind of analysis has not been reported.The results presented here extend our knowledge of how S. oneidensis catalyzes the reduction of insoluble substrates. Experiments using a model iron chelator and electron shuttle are consistent with electron shuttling being the primary mechanism by which flavins enhance insoluble iron oxide reduction rates. Moreover, we demonstrate that MR-1 reduces extracellular flavins at physiologically relevant rates and that the Mtr pathway accounts for at least 95% of this activity. The specific activities of various mutant strains lacking Mtr pathway components on poised-potential electrodes also are reported. Our data suggest that MtrC is responsible for most of the electron transfer to carbon electrodes, while OmcA is involved in attachment and has a lesser role in electron transfer. These observations could have broader implications regarding the role of OmcA in the reduction of soluble and insoluble substrates (8, 9, 36).     

Conformation dependence of the CαDα stretch mode in peptides: Side‐chain influence in dipeptide structures

We have shown by theoretical studies of alanine peptides that the C^αD^α stretch frequency could be particularly useful for determining peptide structure because of its sensitivity to the φ,ψ torsion angles at the C^α atom. To demonstrate that this is a robust methodology worthy of experimental exploration, we have also shown that this mode is even more determinative of conformation in aqueous solution, mainly as a result of the development of differential C^α? D^α···O(water) interactions. As further assurance, we now determine the influence of the side chain on this band, showing for aliphatic, a polar, and an aromatic side chains that the dependence is minor and explaining why this is also expected for other side chains. These results should stimulate new experimental methodologies in the field of peptide structure determination. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1065–1071, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor induction

The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h^?1 with an initial cell mass of less than 0.6 OD₆₀₀. Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9 OD₆₀₀ or higher, or at low dilution rates (<0.05 h^?1) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states. Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron‐supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6 h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron‐limited chemostat cultures was observed compared to iron‐supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology. Biotechnol. Bioeng. 2010;105: 955–964. © 2009 Wiley Periodicals, Inc.     

Wealth transmission and inequality among hunter-gatherers

We report quantitative estimates of intergenerational transmission and population-wide inequality for wealth measures in a set of hunter-gatherer populations. Wealth is defined broadly as factors that contribute to individual or household well-being, ranging from embodied forms such as weight and hunting success to material forms such household goods, as well as relational wealth in exchange partners. Intergenerational wealth transmission is low to moderate in these populations, but is still expected to have measurable influence on an individual's life chances. Wealth inequality (measured with Gini coefficients) is moderate for most wealth types, matching what qualitative ethnographic research has generally indicated (if not the stereotype of hunter-gatherers as extreme egalitarians). We discuss some plausible mechanisms for these patterns, and suggest ways in which future research could resolve questions about the role of wealth in hunter-gatherer social and economic life.