Cyanidin 3-malonylglucoside in Cichorium intybus

The major anthocyanin of red leaves of Cichorium intybus has been identified as cyanidin 3-O-β-(6-O-malonyl)-d-glucopyranoside by fast atom bombardment mass spectrometry and NMR spectroscopy.     

Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer

Background  

Uncovering the molecular mechanism underlying expansion of hematopoietic stem and progenitor cells is critical to extend current therapeutic applications and to understand how its deregulation relates to leukemia. The characterization of genes commonly relevant to stem/progenitor cell expansion and tumor development should facilitate the identification of novel therapeutic targets in cancer.     

Impact of Nosema (Microsporidia) infection and fumagillin treatment on Lygus lineolaris (Hemiptera: Miridae)

A consistent supply of healthy tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is necessary for the development of novel management strategies targeting this pest. After being in culture for several years, a substantial portion of a tarnished plant bug colony was found to be infected with a Nosema (Microsporidia) species. Studies were subsequently undertaken to evaluate the impact of Nosema infection on tarnished plant bug productivity and to test the efficacy of fumagillin to treat this infection. Using buffalo black stain, infections could not be reliably detected in adult tarnished plant bugs until adults were 6-8days post eclosion. Nosema infections reduced adult longevity and fecundity. Maximum fecundity was restored using a concentration of 16.8ppm fumagillin while maximum longevity for females was at a concentration of 33.6ppm fumagillin incorporated into the tarnished plant bug diet. Minimum infection scores were obtained at 67.2ppm, the highest concentration tested. A field survey of tarnished plant bugs in Mississippi found Nosema infections in 3% of wild tarnished plant bugs.     

Interactions of selenium hyperaccumulators and nonaccumulators during cocultivation on seleniferous or nonseleniferous soil--the importance of having good neighbors

? This study investigated how selenium (Se) affects relationships between Se hyperaccumulator and nonaccumulator species, particularly how plants influence their neighbors' Se accumulation and growth. ? Hyperaccumulators Astragalus bisulcatus and Stanleya pinnata and nonaccumulators Astragalus?drummondii and Stanleya?elata were cocultivated on seleniferous or nonseleniferous soil, or on gravel supplied with different selenate concentrations. The plants were analyzed for growth, Se accumulation and Se speciation. Also, root exudates were analyzed for Se concentration. ? The hyperaccumulators showed 2.5-fold better growth on seleniferous than on nonseleniferous soil, and up to fourfold better growth with increasing Se supply; the nonaccumulators showed the opposite results. Both hyperaccumulators and nonaccumulators could affect growth (up to threefold) and Se accumulation (up to sixfold) of neighboring plants. Nonaccumulators S.?elata and A.?drummondii accumulated predominantly (88-95%) organic C-Se-C; the remainder was selenate. S.?elata accumulated relatively more C-Se-C and less selenate when growing adjacent to S.?pinnata. Both hyperaccumulators released selenocompounds from their roots. A.?bisulcatus exudate contained predominantly C-Se-C compounds; no speciation data could be obtained for S.?pinnata. ? Thus, plants can affect Se accumulation in neighbors, and soil Se affects competition and facilitation between plants. This helps to explain why hyperaccumulators are found predominantly on seleniferous soils.     

Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats/min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.     

Analysis of proline reduction in the nosocomial pathogen Clostridium difficile

Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.     

Presence of Coxiella burnetii DNA in the Environment of the United States, 2006 to 2008

Coxiella burnetii is an obligate intracellular bacterium that causes the zoonotic disease Q fever. Because C. burnetii is highly infectious, can survive under a variety of environmental conditions, and has been weaponized in the past, it is classified as a select agent and is considered a potential bioweapon. The agent is known to be present in domestic livestock and in wild animal populations, but the background levels of C. burnetii in the environment have not been reported. To better understand the amount of C. burnetii present in the environment of the United States, more than 1,600 environmental samples were collected from six geographically diverse parts of the United States in the years 2006 to 2008. DNA was purified from these samples, and the presence of C. burnetii DNA was evaluated by quantitative PCR of the IS1111 repetitive element. Overall, 23.8% of the samples were positive for C. burnetii DNA. The prevalence in the different states ranged from 6 to 44%. C. burnetii DNA was detected in locations with livestock and also in locations with primarily human activity (post offices, stores, schools, etc.). This study demonstrates that C. burnetii is fairly common in the environment in the United States, and any analysis of C. burnetii after a suspected intentional release should be interpreted in light of these background levels. It also suggests that human exposure to C. burnetii may be more common than what is suggested by the number of reported cases of Q fever.The Gram-negative obligate intracellular bacterium Coxiella burnetii can infect humans and cause Q fever, an acute febrile illness (15, 17). Most cases of Q fever have fairly nonspecific symptoms, such as high fever, headache, myalgia, cough, and fatigue (29). Over one-third of patients may show signs of pneumonia or hepatitis (17). Acute cases typically resolve in 1 to 2 weeks, but a small percentage of Q fever cases result in a chronic infection that can present as endocarditis and be life-threatening (12).Q fever occurs worldwide, and numerous natural outbreaks have been reported in the United States (2, 23, 25) and other countries (5, 11, 18, 20, 22, 24). An ongoing natural outbreak in the Netherlands resulted in more than 2,000 cases of Q fever from 2007 to 2009 (27). In the United States Q fever became a nationally notifiable disease in 1999, and increasing numbers of cases have been reported to the CDC in recent years. However, the highest number of annual cases in the United States so far has been 171, reported in 2007 (8). Although this is a fairly small number of reported cases, it is possible that the number of actual cases in the United States is much higher. The relatively nonspecific nature of Q fever symptoms makes the disease difficult to diagnose, and people infected with C. burnetii are likely to show a diversity of symptoms with variable severity. The idea that Q fever is underreported is supported by our recent data using serum samples from the National Health and Nutrition Examination Survey (NHANES) to determine that the seroprevalence in the United States among people who are ≥20 years old is 3.1% (1).A common mechanism for people to become infected with C. burnetii is the inhalation of aerosolized bacteria. Potential sources for aerosolized C. burnetii are livestock and other animals. It is known that many herds of livestock are infected with C. burnetii and that seroprevalence rates in a variety of wild animal species can be quite high (17). Infected livestock herds do not typically show clinical signs of infection, but surges in abortion rates have been reported, particularly with goats (9, 10, 17). It is known that C. burnetii can replicate to high levels in the placenta of infected animals and that infectious C. burnetii can be spread to humans during parturition (9). The prevalence of C. burnetii in animals makes contact with animals a likely risk factor for Q fever. For example, the ongoing Q fever outbreak in the Netherlands has been linked to Q fever infections in goat farms (27), and we have recently found that 22.2% of a group of 508 veterinarians had antibodies against C. burnetii, a much higher seroprevalence than in the general U.S. population (31).C. burnetii exists as a replicating large-cell variant (LCV), but nonreplicating bacteria can form a more stable small-cell variant (SCV) (4). Although it is not an endospore, the SCV form of Coxiella is known to be very stable under a variety of conditions (16). C. burnetii is also highly infectious, with a dose of 1 to 10 organisms capable of causing Q fever in humans (30). These unique features of C. burnetii, along with its aerosol route of transmission, have led to the designation of C. burnetii as a category B bioterrorism weapon and inclusion on the list of select agents. The potential for the use of C. burnetii as a bioweapon was explored in detail by the U.S. bioweapons program of the 1950s and 1960s (26). Although not typically lethal, C. burnetii is considered a threat due to its ability to cause widespread debilitating illness. Indeed, many U.S. soldiers returning from Iraq between 2005 and 2008 suffered from Q fever while deployed (6, 7). These cases are suspected to be naturally acquired infections.The potential for both intentional releases and natural outbreaks makes it important to understand the presence of C. burnetii in the environment. Investigations of the source of Q fever cases will include a determination of the presence of C. burnetii in the environment from which the bacteria may have been acquired. The purpose of this study was to analyze a large number of samples across a wide geographic distribution in the United States and to establish a baseline for the presence of C. burnetii in different regions of the country.     

Estimation of the Initial Viral Growth Rate and Basic Reproductive Number during Acute HIV-1 Infection

During primary infection, the number of HIV-1 particles in plasma increases rapidly, reaches a peak, and then declines until it reaches a set point level. Understanding the kinetics of primary infection, and its effect on the establishment of chronic infection, is important in defining the early pathogenesis of HIV. We studied the viral dynamics of very early HIV-1 infection in 47 subjects identified through plasma donation screening. We calculated how fast the viral load increases and how variable this parameter is among individuals. We also estimated the basic reproductive ratio, the number of new infected cells generated by an infectious cell at the start of infection when target cells are not limiting. The initial viral doubling time had a median of 0.65 days with an interquartile range of 0.56 to 0.91 days. The median basic reproductive ratio was 8.0 with an interquartile range of 4.9 to 11. In 15 patients, we also observed the postpeak decay of plasma virus and found that the virus decay occurred at a median rate of 0.60 day⁻¹, corresponding to a half-life of 1.2 days. The median peak viral load was 5.8 log₁₀ HIV-1 RNA copies/ml, and it was reached 14 days after the virus was quantifiable with an assay, with a lower limit of detection of 50 copies/ml. These results characterize the early plasma viral dynamics in acute HIV infection better than it has been possible thus far. They also better define the challenge that the immune response (or therapeutic intervention) has to overcome to defeat HIV at this early stage.During primary infection, the number of human immunodeficiency virus type 1 (HIV-1) particles in plasma increases rapidly, reaches a peak, and then declines until it reaches a set point level (i.e., a quasi-steady state) (3, 26). Often, the peak in viral load coincides with the first appearance of an acquired immune response. Thus, early HIV infection can be seen as a race between the immune system and the virus (4). It has been suggested, based on the macaque model with simian immunodeficiency virus (SIV) infection, that early viral expansion is somewhat homogeneous across subjects, but the viral load set point varies by orders of magnitude (14). However, studies with SIV also suggest that the early events during viral expansion, i.e., before the peak, are important in defining the viral load set point later in infection (14).Thus, improved knowledge of the very early expansion of HIV-1 will be beneficial for our understanding of primary infection and its effect on the establishment of chronic infection. Moreover, if the immune system primed by a vaccine could respond quickly enough to HIV, perhaps it would be possible to prevent infection. However, all but the recombinant canarypox-gp120 vaccine, used in the RV144 trial in Thailand (23), have failed to provide protection, and the immune response generated by T-cell-based vaccines has been described as “too little, too late” (1, 5, 24). Here, we characterize the early events in infection and the prepeak expansion of HIV-1 to better understand the biology of infection.We examine longitudinal viral load data from 47 frequent plasma donors who became HIV positive during the course of their plasma donations. Thus, this data set includes samples in which virus was absent or below the limit of detection of the assay used, as well as viral loads at very early times postexposure with HIV. From this data, we quantify the rate of viral expansion during primary HIV infection. Previously, Fiebig et al. (8, 9) analyzed some aspects of early viral load expansion, the existence of viral blips, and the timing of HIV-1 marker expression, defining the stages for early infection (9). Here, we extend these analyses to characterize in detail the expansion of the virus and its basic reproductive ratio, R₀. In the context of host viral dynamics, R₀ is a measure of whether a virus can establish infection (12). It specifically measures how many cells a single infected cell will infect when there is no target cell limitation. If R₀ is less than 1, on average an infected cell will infect less than 1 susceptible cell, and the infection will die out. If R₀ is greater than 1, on average an infected cell will infect more than 1 susceptible cell, and generally the infection will spread (25).R₀ for HIV infection in humans has been estimated previously in smaller data sets. Little et al. (15) used both viral load and CD4⁺ T-cell count data to find R₀ for four individuals whose infection was identified within a couple of weeks of exposure. Stafford et al. (27) estimated R₀ from viral load data obtained from 10 primary infection patients, again identified within a few weeks of infection. Our work differs from these previous studies in that we analyzed a much larger number of patients and the viral load data that we analyzed encompassed the earliest stages of infection, in most cases before the viral load was even detectable. By contrast, both Little et al. (15) and Stafford et al. (27) analyzed data obtained primarily near or after the peak in viral load.     

Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT.Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca²⁺ imaging and [³H]ryanodine binding.A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca²⁺ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine.The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.Key words: arrhythmia, calcium, death sudden, genetics, ion channels