Autofertility and rate of cross-fertilization: crucial characters for breeding synthetic varieties in faba beans (Vicia faba L.)

Summary To increase the level and stability of yield in faba beans (Vicia faba L.), heterosis must be exploited. Hybrids are not available because of the instability of male sterility. Synthetic varieties can and should be bred. Thus, we studied the reproductive behavior of this partially allogamous, insect-pollinated crop. Autofertility (AF) and the rate of cross-fertilization (C) were measured in 36 inbred lines and 28 crosses in F₁, F₂, and F₃ generations for 3 years at Hohenheim, Stuttgart, FRG. Heterozygosity strongly increased AF and decreased C. AF was negatively correlated with C. AF varied from 1% to 98%, and C varied from 7% to 82%. Heritability for both characters was high. For an optimum exploitation of heterosis, breeders should utilize lines with high C as variety components. It is often labor-intensive to multiply such lines, due to low AF. Hence, breeders tend to use autofertile lines with rather limited C. We showed that even in this case about 50% of the total heterosis, which equals a yield increase of at least 25% over the inbred line level, is realized. An increase in yield stability due to heterogeneity will occur simultaneously.     

Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip

INTRODUCTION: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. METHODS: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer. RESULTS: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used. CONCLUSIONS: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.     

On the mechanisms of arrhythmias in the myocardium of mXinalpha-deficient murine left atrial-pulmonary veins

We have previously shown that left atrial-pulmonary vein tissue (LA-PV) can generate reentrant arrhythmias (atrial fibrillation, AF) in wild-type (mXinalpha+/+) but not in mXinalpha-null (mXinalpha-/-) mice. With the present experiments, we investigated the arrhythmogenic activity and the underlying mechanisms in mXinalpha+/+ vs. mXinalpha-/- LA-PV. Electrical activity and conduction velocity (CV) were recorded in LA-PV by means of a MED64 system. CV was significantly faster in mXinalpha+/+ than in mXinalpha-/- LA-PV and it was increased by 1 muM isoproterenol (ISO). AF could be induced by fast pacing in the mXinalpha+/+ but not in mXinalpha-/- LA-PV where automatic rhythms could occur. ISO increased the incidence of AF in Xinalpha+/+ whereas it increased that of automatic rhythms in mXinalpha-/- LA-PV. In LA-PV with the right atrium attached (RA-LA-PV), automatic rhythms occurred in all preparations. In mXinalpha+/+ RA-LA-PV simultaneously treated with ISO, strophanthidin and atropine, the incidence of the automatic rhythm was about the same, but AF increased significantly. In contrast, in mXinalpha-/- RA-LA-PV under the same condition, the automatic rhythm was markedly enhanced, but still no AF occurred. Conventional microelectrode techniques showed a longer APD(90) and a less negative maximum diastolic potential (MDP) in mXinalpha-/- than mXinalpha+/+ LA-PV tissues. Whole-cell current clamp experiments also showed a less negative MDP in mXinalpha-/- vs. mXinalpha+/+ LA-PV cardiomyocytes. The fact that AF could be induced by fast pacing under several conditions in mXinalpha+/+ but not in mXinalpha-/- LA-PV preparations appears to be due to a slower CV, a prolonged APD(90), a less negative MDP and possibly larger areas of conduction block in mXinalpha-/- myocardial cells. In contrast, the non-impairment of automatic and triggered rhythms in mXinalpha-/- preparations may be due to the fact that the mechanisms underlying these rhythms do not involve cell-to-cell conduction.     

Computational Mapping Identifies Localized Mechanisms for Ablation of Atrial Fibrillation

Atrial fibrillation (AF) is the most common heart rhythm disorder in the Western world and a common cause of hospitalization and death. Pharmacologic and non-pharmacologic therapies have met with limited success, in part due to an incomplete understanding of the underlying mechanisms for AF. AF is traditionally characterized by spatiotemporally disorganized electrical activation and, although initiating triggers for AF are described, it is unclear whether AF is sustained by spatially meandering continuous excitation (re-entrant waves), localized electrical sources within the atria, or some other mechanism. This has limited therapeutic options for this condition. Here we show that human AF is predominantly caused by a small number (1.8±0.9) of localized re-entrant waves or repetitive focal beats, that remain stable with limited spatial migration over prolonged periods of time. Radiofrequency ablation that selectively targeted the sites of these sources was able to immediately terminate fibrillation and eliminate the arrhythmia with high success. Our results show that human AF, despite apparent spatiotemporal disorganization, is often perpetuated by a few spatially-constrained and temporally conserved sources whose targeted ablation can eliminate this complex rhythm disorder.     

Circ-U2AF1 promotes human glioma via derepressing neuro-oncological ventral antigen 2 by sponging hsa-miR-7-5p

The prognosis for human glioma, a malignant tumor of the central nervous system, is poor due to its rapid growth, genetic heterogeneity, and inadequate understanding of its underlying molecular mechanisms. Circular RNAs composed of exonic sequences, represent an understudied form of noncoding RNAs (ncRNAs) that was discovered more than a decade ago, function as microRNA sponges. We aimed to assess the relationship between circ-U2AF1 (CircRNA ID: hsa_circ_0061868) and hsa-mir-7-5p and examine their effects on proliferation, apoptosis, and the metastatic phenotype of glioma cells regulated by neuro-oncological ventral antigen 2 (NOVA2). We found that the expression levels of circ-U2AF1 and NOVA2 were upregulated, while hsa-miR-7-5p was downregulated in human glioma tissues and glioma cell lines. Our data and bioinformatic analysis indicated the association of these molecules with glioma grade, a positive correlation between circ-U2AF1 and NOVA2 expression levels and a negative correlation of hsa-miR-7-5p with both circ-U2AF1 and NOVA2, respectively. In addition, silencing of circ-U2AF1 expression resulted in increased hsa-miR-7-5p expression and decreased NOVA2 expression both in vitro and in vivo. Luciferase assay confirmed hsa-miR-7-5p as a direct target of circ-U2AF1 and NOVA2 as a direct target of hsa-miR-7-5p. Functionally, silencing of circ-U2AF1 inhibits glioma development by repressing NOVA2 via upregulating hsa-miR-7-5p both in vitro and in vivo. Thus, we assumed that circ-U2AF1 promotes glioma malignancy via derepressing NOVA2 by sponging hsa-miR-7-5p. Taken together, we suggest that circ-U2AF1 can be a prognostic biomarker and the circ-U2AF1/hsa-miR-7-5p/NOVA2 regulatory pathway may be a novel therapeutic target for treating gliomas.     

Characterization of U2AF6, a Splicing Factor Related to U2AF35

The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. U2AF(35) has multiple functions in pre-mRNA splicing. First, U2AF(35) has been shown to function by directly interacting with the AG at the 3' splice site. Second, U2AF(35) is thought to play a role in the recruitment of U2AF(65) by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF(35) and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF(35), designated U2AF(26). The N-terminal 187 amino acids of U2AF(35) and U2AF(26) are nearly identical. However, the C-terminal domain of U2AF(26) lacks many characteristics of the U2AF(35) RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF(26) can associate with U2AF(65) and can functionally substitute for U2AF(35) in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF(26) functions by enhancing the binding of U2AF(65) to weak 3' splice sites. These studies identify U2AF(26) as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF(35), U2AF(26) may play a role in regulating alternative splicing.     

Frequency analysis of atrial fibrillation from the surface electrocardiogram

Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. Neither the natural history of AF nor its response to therapy are sufficiently predictable by clinical and echocardiographic parameters. Atrial fibrillatory frequency (or rate) can reliably be assessed from the surface electrocardiogram (ECG) using digital signal processing (filtering, subtraction of averaged QRST complexes, and power spectral analysis) and shows large inter-individual variability. This measurement correlates well with intraatrial cycle length, a parameter which appears to have primary importance in AF domestication and response to therapy. AF with a low fibrillatory rate is more likely to terminate spontaneously, and responds better to antiarrhythmic drugs or cardioversion while high rate AF is more often persistent and refractory to therapy. In conclusion, frequency analysis of AF seems to be useful for non-invasive assessment of electrical remodeling in AF and may subsequently be helpful for guiding AF therapy.     

Effects of the antlered form of Ganoderma lucidum on tumor growth and metastasis in cyclophosphamide-treated mice

We examined the alleviation of cyclophosphamide-induced immunodepression by the antlered form of Ganoderma lucidum (G. lucidum AF) and also evaluated the anti-tumor and anti-metastatic effects of G. lucidum AF in cyclophosphamide-treated mice. G. lucidum AF alleviated cyclophosphamide-induced decrease in body weight, natural killer (NK) activity, interferon (IFN)-gamma production, and cytotoxic T lymphocyte (CTL) activity, and inhibited the abnormal increase and decrease in interleukine (IL)-4 level due to cyclophosphamide administration. Post-treatment with cyclophosphamide and G. lucidum AF significantly inhibited tumor growth in MM 46-bearing mice. When Lewis lung carcinoma cells were injected into mice after a cyclophosphamide administration, metastasis of these cells to the lung was increased, but G. lucidum AF suppressed it. The anti-tumor and anti-metastatic effects of the combination of G. lucidum AF and cyclophosphamide might influence the modulatory effects of G. lucidum AF on both cellular and humoral immunity. These findings suggest that G. lucidum AF would be beneficial in alleviating the reduction of immune response by chemotherapeutic anti-cancer drugs.     

Estimating the prevalence of atrial fibrillation from a three‐class mixture model for repeated diagnoses

Atrial fibrillation (AF) is an abnormal heart rhythm characterized by rapid and irregular heartbeat, with or without perceivable symptoms. In clinical practice, the electrocardiogram (ECG) is often used for diagnosis of AF. Since the AF often arrives as recurrent episodes of varying frequency and duration and only the episodes that occur at the time of ECG can be detected, the AF is often underdiagnosed when a limited number of repeated ECGs are used. In studies evaluating the efficacy of AF ablation surgery, each patient undergoes multiple ECGs and the AF status at the time of ECG is recorded. The objective of this paper is to estimate the marginal proportions of patients with or without AF in a population, which are important measures of the efficacy of the treatment. The underdiagnosis problem is addressed by a three‐class mixture regression model in which a patient's probability of having no AF, paroxysmal AF, and permanent AF is modeled by auxiliary baseline covariates in a nested logistic regression. A binomial regression model is specified conditional on a subject being in the paroxysmal AF group. The model parameters are estimated by the Expectation‐Maximization (EM) algorithm. These parameters are themselves nuisance parameters for the purpose of this research, but the estimators of the marginal proportions of interest can be expressed as functions of the data and these nuisance parameters and their variances can be estimated by the sandwich method. We examine the performance of the proposed methodology in simulations and two real data applications.     

Effects of cooling rate, annealing time and biological antifreeze concentration on thermal hysteresis reading

Thermal hysteresis (TH) readings depend on the cooling rate, annealing time and the concentration of the biological antifreeze (AF) (i.e., antifreeze protein or antifreeze glycoprotein). Such time- and concentration-dependent TH readings are not true (or absolute) values. The true TH should be independent of time and AF concentration, and it should be a unique value for a given AF. Only the true TH can be used to assess the activity of an AF. A mathematical model is proposed to explain the time- and concentration-dependent behavior of AFs. The model assumes a reversible Langmuir adsorption mechanism for the AF molecules and the Kelvin effect to be applicable. A TH equation that correlates the TH reading with the cooling rate, annealing time and AF concentration was derived. The time-dependent behavior was attributed to the slow adsorption process of the AF. The theoretical calculations were compared with previously published data on the effects of the cooling rate, annealing time and AF concentration on TH readings. The calculated results agree qualitatively with the literature data. The experimental methodology required for obtaining the true TH of an AF is suggested.     

Amniotic fluid zinc in risk pregnancies

The zinc concentration of amniotic fluid (AF) of 129 pregnant women was analyzed by the flame atomic absorption spectrometry. This prospective study was performed in order to find out whether the determination of the AF zinc concentration can be used to monitor the growth and development of the fetus. There were two groups of patients: early stage (15th–19th gestation wk) in which the amniocentesis was performed as a prenatal genetic examination, and late stage (26th–40th wk) in which the amniocentesis was performed due to obstetric reasons. The average AF zinc concentrations were 1.2 and 1.0 μmol/L in the early and late gestation group, respectively. The AF zinc concentration did not correlate with the weight, height, or Apgar scores of the newborn nor with the maternal diseases, age, or parity. The AF zinc concentration in the late gestation group was significantly lower if the fetus was male than if it was female. If the AF was greenish the zinc concentration was elevated. One malformation, congenital nephrosis, with an exceptionally high zinc concentration (9.0 μmol/L), was found.     

Effects of cooling rate,annealing time and biological antifreeze concentration on thermal hysteresis reading

Thermal hysteresis (TH) readings depend on the cooling rate, annealing time and the concentration of the biological antifreeze (AF) (i.e., antifreeze protein or antifreeze glycoprotein). Such time- and concentration-dependent TH readings are not true (or absolute) values. The true TH should be independent of time and AF concentration, and it should be a unique value for a given AF. Only the true TH can be used to assess the activity of an AF. A mathematical model is proposed to explain the time- and concentration-dependent behavior of AFs. The model assumes a reversible Langmuir adsorption mechanism for the AF molecules and the Kelvin effect to be applicable. A TH equation that correlates the TH reading with the cooling rate, annealing time and AF concentration was derived. The time-dependent behavior was attributed to the slow adsorption process of the AF. The theoretical calculations were compared with previously published data on the effects of the cooling rate, annealing time and AF concentration on TH readings. The calculated results agree qualitatively with the literature data. The experimental methodology required for obtaining the true TH of an AF is suggested.     

Antagonist-induced, activation function-2-independent estrogen receptor alpha phosphorylation

Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.     

Atrial fibrillation in β-thalassemia patients with a focus on the role of iron-overload and oxidative stress: A review

Cardiac complications including arrhythmia and especially atrial fibrillation (AF) are common causes of death in β-thalassemia patients. The main factor in the etiopathogenesis of these complications is iron overload, which results in increased oxidative stress. Although there is a known association between cardiac complications and iron overload in β-thalassemia patients, there is no comprehensive review on AF and excessive iron with a focus on oxidative stress in these patients. The aim of this article was to review the different aspects of AF in β-thalassemia patients with a focus on the prevention and treatment of AF by using iron chelators and/or anti-oxidants. AF in β-thalassemia patients is more common than in the general population. One of the most important causes of AF is cardiac iron overload and the harmful effects of increased oxidative stress. Iron-induced AF can be reversed by using an intensive iron chelation regimen. Based on a few experimental studies, the combination of iron chelators with some anti-oxidants, including NAC, vitamin C, and acetaminophen, can lead to improved cardiac protection. However, the effect of such combinations on cardiac arrhythmias should be further evaluated with animal and human studies.     

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.     

Wide home ranges for widely foraging lizards

Space usage by animals may be influenced by a range of factors. In this study we investigate whether foraging behaviour affects the home range size of lizards. Two distinct tactics of foraging have been recognized in predators: sit-and-wait foraging (SW) and active foraging (AF). Foraging activity level of a data set of lizard species, mainly compiled from literature, is compared with their home range sizes. Two opposite predictions can be made about foraging in connection with home range area: on the one hand, SW species may exhibit larger home ranges due to their mating system; on the other hand, AF species have higher metabolic energy and thus food requirements and can be expected to have larger home ranges that have to yield this food. This study shows that percentage of the time moving (as an index of foraging mode) correlates positively with home range, even after correcting for body mass, and these patterns remain when phylogenetic relationships are taken into account. We thus conclude that home range areas parallel activity levels in lizards.     

Isolation of a Factor from Apple that Agglutinates Erwinia amylovora

Extracts prepared from apple seeds contain a factor (AF) capable of agglutinating cells of Erwinia amylovora. In drop agglutination tests, AF is more active in agglutinating an avirulent, acapsular strain of E. amylovora than a virulent, capsular strain. AF precipitates in agar plates with a receptor derived from boiled cells of avirulent acapsular strain and, therefore, can be located during fractionation by rocket electrophoresis. AF was heat-stable and had a pH optimum for agglutination near congruent with3.6 pH. The agglutination activity was not affected by the presence of Mg(2+), Ca(2+), or EDTA. AF was separated into two fractions (AF I and AF II) by elution from a Bio-Gel P-100 column. The precipitin and agglutination activities associated with AF II were found to be present in a positively charged molecule which was sensitive to treatment with protease and trypsin and, hence, presumably resides in a protein. The approximate molecular weight of AF II was determined to be 12,600 daltons. Besides precipitating the receptor derived from cells of avirulent acapsular strain, AF II was capable of precipitating extracellular polysaccharide from cultures of virulent capsular strain, sodium polygalacturonate, and carboxymethylcellulose. These three polymers also inhibited the agglutination activity associated with AF II. AF II could be replaced by poly-l-lysines in both the precipitin and agglutination assays. In addition, in antigen absorption experiments, poly-l-lysines were found to remove the receptors for AF II from the boiled extracts of avirulent acapsular strain. Based on these observations, it is proposed that the activity of AF II resides in a highly positively charged protein which causes agglutination of bacterial cells by interacting on a charge-charge basis with negatively charged components on the surface of the bacterial cells.     

A review on sample entropy applications for the non-invasive analysis of atrial fibrillation electrocardiograms

The application of non-linear metrics to physiological signals is a valuable tool because “hidden information” related to underlying mechanisms can be obtained. In this respect, approximate entropy (ApEn) is the most popular non-linear regularity index that has been applied to physiological time series. However, ApEn presents some shortcomings, such as bias, relative inconsistency and dependence on the sample length. A modification of ApEn, named sample entropy (SampEn), was introduced to overcome these deficiencies. Recently, in the context of electrocardiography, SampEn has been applied to study non-invasively atrial fibrillation (AF), which is the most common arrhythmia encountered in clinical practice with unknown mechanisms provoking its onset and termination. Useful clinical information, that could help for a better understanding of AF mechanisms, has been obtained through the application of SampEn to electrocardiographic (ECG) recordings. This work reviews its application in the context of non-invasive analysis of AF. During this arrhythmia, atrial and ventricular components can be regarded as unsynchronized activities, whereby, the application of SampEn to the analysis of each component will be described separately. In first place, clinical challenges in which SampEn has been successfully applied to estimate AF organization from the atrial activity pattern are presented. The AF organization study can provide information on the number of active reentries, which can help to improve AF treatment and to take the appropriate decisions on its management. Next, the heart rate variability study via SampEn, to characterize ventricular response and predict AF onset, is described. Through the aforementioned applications it is remarked throughout this review that SampEn can be considered as a very promising and useful tool towards the non-invasive understanding of AF.