Success of maximum likelihood phylogeny inference in the four-taxon case

We used simulated data to investigate a number of properties of maximum- likelihood (ML) phylogenetic tree estimation for the case of four taxa. Simulated data were generated under a broad range of conditions, including wide variation in branch lengths, differences in the ratio of transition and transversion substitutions, and the absence of presence of gamma-distributed site-to-site rate variation. Data were analyzed in the ML framework with two different substitution models, and we compared the ability of the two models to reconstruct the correct topology. Although both models were inconsistent for some branch-length combinations in the presence of site-to-site variation, the models were efficient predictors of topology under most simulation conditions. We also examined the performance of the likelihood ratio (LR) test for significant positive interior branch length. This test was found to be misleading under many simulation conditions, rejecting too often under some simulation conditions. Under the null hypothesis of zero length internal branch, LR statistics are assumed to be asymptotically distributed chi 2(1); with limited data, the distribution of LR statistics under the null hypothesis varies from chi 2(1).      

Establishment of a doxycycline-regulated cell line with inducible,doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells

p53 is important in the development of hepatocellular carcinoma (HCC) and in therapeutic approaches, but the mechanism whereby it inhibits HCC growth is still unclear. The aim of the present study was to establish a HCC cell system in which p53 levels can be regulated. Full-length wild-type p53 cDNA obtained by PCR was cloned into a retroviral response vector controlled by the tetracycline responsive element (RevTRE-p53). The regulatory vectors RevTet-Off and RevTRE-p53 were transfected into a packaging cell line, PT67. Hep3B cells in which the p53 gene was deleted were infected with RevTet-Off viral particles from the PT67. Three G418-resistant cell clones with high luciferase expression and low background were infected with RevTRE-p53. By screening dozens of RevTRE-p53-infected clones with hygromycin we identified the one with the highest expression of p53 and the lowest background after doxycycline treatment. The results showed that p53 expression in this cell clone could be simply turned on or off by removing or adding doxycycline. Furthermore, it was found that the level of p53 protein was negatively and sensitively related to the doxycycline concentration. In conclusion, we have established a HCC cell line in which p53 expression can be switched on or off and regulated in a dose- and time-dependent manner.     

Differential expression of genes coding for EGF-like factors and ADAMTS1 following gonadotropin stimulation in normal and transformed human granulosa cells

We have demonstrated previously that the synthesis of epiregulin and amphiregulin, of the EGF-like growth factor family, is stimulated by luteinizing hormone in human follicular (granulosa) cells obtained from in vitro fertilization program. In the present work, we demonstrate that H89, a PKA inhibitor, attenuated the expression of these growth factors both in the mRNA and the protein levels, suggesting PKA involvement in this signaling pathway. SV40-transformed human granulosa cells showed higher basal levels of epiregulin and amphiregulin than normal cells, which were still elevated following cAMP stimulation by Forskolin. Cleavage by a disintegrin and metalloproteinases (ADAMs) is essential for activation of these growth factors, allowing their interaction with EGF receptor. Expression of ADAMTS1 and ADAM12 was downregulated by cAMP in normal, but not in SV40-transformed cells, suggesting that in normal cells epiregulin and amphiregulin activity is downregulated by a feedback mechanism that may be lost in SV40-transformed cells and their loss of downregulation may be involved in the development of ovarian tumors.     

A pacemaker cell pair model based on the phase response curve

A pacemaker cell pair model and the dynamic interaction between the two pacemaker cells is described in this paper. It is an extension of our single pacemaker cell model, in which we studied its response to repetitive external depolarization stimulations. This model is a simple model based on the two most important functional properties of the cardiac pacemaker cells: its intrinsic pacemaker cycle length, which is an `internal' parameter of the cell, and the phase response curve (PRC), which is an `overall collective' function. The PRC contains all the `information' about the possible interactions of the pacemaker cell with the outside world (interaction with surrounding cells, external stimulus, etc.). First, we examined the properties and solutions of 1:1 synchronization between two pacemaker cells. We found that in order to achieve synchronization between two pacemaker cells, there should be limitations on the PRC parameters, which depend on the cells intrinsic cycle lengths. Next, we investigated the 2:1 entrainment state between two interacting pacemaker cells. We found that there is not necessarily a unique solution for this state as there was for the 1:1 state. Finally, we ran our computer model to investigate the properties of more complex patterns of entrainment between two pacemaker cells. As a result of our analytical study, we unveil two new important parameters, which are fully defined as a function of the PRC parameters: (1) the `accelerator factor' which describes the tendency of a pair of interacting pacemaker cells to synchronize at a common cycle length, which is closer to the faster cycle of the pair; (2) the `degree of coupling', which describes the range of the 1:1 synchronization and the `strength' of the interaction between a pair of interacting pacemaker cells. Those two interaction parameters arise as helpful `tools' for the understanding of synchronization and mutual entrainment mechanisms between pacemaker cells. Therefore, this study establishes the PRC as an important determinant and a useful approach for the understanding of the dynamic interaction of pacemaker cells among themselves and with the outside world. Received: 12 May 1997 / Accepted in revised form: 22 April 1998     

Detecting substitution-rate heterogeneity among regions of a nucleotide sequence

Likelihood-ratio statistics are proposed to test for heterogeneity in nucleotide substitution rate among regions of a DNA sequence. The tests examine three-sequence phylogenies, and two specific tests are proposed: a test to detect rate heterogeneity among genic regions within a sequence, over all evolutionary lineages; and a test to detect rate heterogeneity among regions in a specific evolutionary lineage. Simulations examine the ability of tests to detect a single region that varies in nucleotide substitution rate relative to the remainder of the sequence. A 50-bp region with a fivefold substitution-rate increase can be detected > or = 90% of the time when it is found in all three lineages of the phylogeny, and a 50-bp region of fivefold rate increase can be detected with approximately 70% power when it is found in only one evolutionary lineage. Simulation also examines the effect of transition- and transversion-rate differences. The tests are applied to published DNA sequences. While the tests are powerful, significant results can be difficult to interpret biologically.      

Predictors of mortality of patients newly diagnosed with clinical type 2 diabetes: a 5-year follow up study

Background

At diabetes diagnosis major decisions about life-style changes and treatments are made based on characteristics measured shortly after diagnosis. The predictive value for mortality of these early characteristics is widely unknown. We examined the predictive value of patient characteristics measured shortly after diabetes diagnosis for 5-year all-cause and cardiovascular mortality with special reference to self-rated general health.

Methods

Data were from a population-based sample of 1,323 persons newly diagnosed with clinical diabetes and aged 40 years or over. Possible predictors of mortality were investigated in Cox regression models.

Results

Multivariately patients who rated their health less than excellent experienced increased all-cause and cardiovascular mortality. These end-points also increased with sedentary life-style, relatively young age at diagnosis and presence of cardiovascular disease (CVD) at diagnosis. Further predictors of all-cause mortality were male sex, low body mass index and cancer, while cardiovascular mortality increased with urinary albumin concentration.

Conclusions

We found that patients who rated their health as less than excellent had increased 5-year mortality, similar to that of patients with prevalent CVD, even when biochemical, clinical and life-style variables were controlled for. This finding could motivate doctors to discuss perceptions of health with newly diagnosed diabetic patients and be attentive to patients with suboptimal health ratings. Our findings also confirm that life-style changes and optimizing treatment are particularly relevant for relatively young and inactive patients and those who already have CVD or (micro)albuminuria at the time of diabetes diagnosis.     

Decrease in anaerobe-related bacteraemias and increase in Bacteroides species isolation rate from 1998 to 2007: A retrospective study

Conflicting data have accumulated in recent years regarding the incidence of anaerobic bacteraemias. The aim of this study was to determine the prevalence of bacteraemias due to anaerobic bacteria and evaluate the importance of anaerobic blood cultures in a university hospital in Israel. A retrospective survey which focused on anaerobic blood culture bottles was performed on blood cultures received in our laboratory during the decade from January 1998 to December 2007. Anaerobic-related bacteraemias decreased during that period, whereas a significant increase was observed in Bacteroides species isolated from the blood cultures (from 18% during 1998–2002 to 43% during 2003–2007). Comparison of the medical records of 54 patients with Bacteroides-related bacteraemia during the two end periods (1998–1999 and 2006–2007) revealed a marked increase in complex underlying diseases. Hypertension and diabetes mellitus type II were found in 29% of the patients in 1998–1999 and increased to 43–45% of the patients in 2006–2007. Ischemic heart disease also increased from 14% of the patients in 1998–1999 to 43% in 2006–2007. We conclude that although positive anaerobic blood cultures account for a small percentage of positive blood samples, the growing involvement of Bacteroides species-related bacteraemias together with an increase in complex underlying diseases in these patients emphasize the importance of anaerobic blood cultures, particularly in patients with co-morbidities.     

Age-related accumulation of pentosidine in aggrecan and collagen from normal and degenerate human intervertebral discs

During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1-A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol x (g of protein)(-1) x year(-1). Using previously reported protein turnover rate constants (k(T)) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (k(F)) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009-13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81+/-0.25 compared with 3.71+/-0.26 micromol of pentosidine x (mol of lysine)(-1) x year(-1) respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year(-1) compared with 0.134 year(-1) for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective k(F) values of 1.81+/-0.25 and 3.18+/-0.37 micromol of pentosidine.(mol of lysine)(-1) x year(-1). We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding.     

Hyper-activated motility in sperm capacitation is mediated by phospholipase D-dependent actin polymerization

In order to fertilize the oocyte, sperm must undergo a series of biochemical changes in the female reproductive tract, known as capacitation. Once capacitated, spermatozoon can bind to the zona pellucida of the egg and undergo the acrosome reaction (AR), a process that enables its penetration and fertilization of the oocyte. Important processes that characterize sperm capacitation are actin polymerization and the development of hyper-activated motility (HAM). Previously, we showed that Phospholipase D (PLD)-dependent actin polymerization occurs during sperm capacitation, however the role of this process in sperm capacitation is not yet known. In the present study, we showed for the first time the involvement of PLD-dependent actin polymerization in sperm motility during mouse and human capacitation. Sperm incubated under capacitation conditions revealed a time dependent increase in actin polymerization and HAM. Inhibition of Phosphatidic Acid (PA) formation by PLD using butan-1-ol, inhibited actin polymerization and motility, as well as in vitro fertilization (IVF) and the ability of the sperm to undergo the AR. The inhibition of sperm HAM by low concentration of butan-1-ol is completely restored by adding PA, further indicating the involvement of PLD in these processes. Furthermore, exogenous PA enhanced rapid actin polymerization that was followed by a rise in the HAM, as well as an increased in IVF rate. In conclusion, our results demonstrate that PLD-dependent actin polymerization is a critical step needed for the development of HAM during mouse and human sperm capacitation.