In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated . Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation. The establishment of successful pregnancy requires a profound reorganization of uterine tissues. Rapid growth and differentiation of the endometrial stroma is the earliest and most striking event in pregnancy. Differentiation of stromal cells leads to the formation of unique cells, termed decidual cells, which differ greatly from the original stromal cells . The dec2α can induce different biological actions at the beginning and at the end of pregnancy. PGF2α, Prostaglandin E2 (PGE2), and 6-keto-PGF1α are produced by the pregnant uterus [2 and PGF2α is observed on day 5 of pregnancy, allowing the decidualization process to take place. When embryo access to the uterus is impaired, production of prostaglandins (PGE2 and PGF2α) is suppressed [2α decreases [2α over certain values can terminate pregnancy [2α is known to induce inhibition of implantation, alteration of embryo development, and induction of luteal regression [2α can induce premature uterine contraction and premature labor [2α secretion occurs, it can alter implantation, induce abortion or even embriolethality [2α levels after implantation can be detrimental for the progression of pregnancy, the aim of this investigation was to determine whether PGF2α affects directly decidual cells and leads to disturbance in the expression of genes crucial for decidual survival.Prostaglandin Ft uterus . An incrppressed . During ecreases . Whereasgression . This rere labor . Dependiethality ,8. BecauPseudopregnancy was induced by mating Holtzman Sprague Dawley female rats with vasectomized male rats. The day a vaginal plug was found was designated day 1 of pseudopregnancy. Decidualization of the uterine endometrium was induced by scratching the antimesometrial surface with a hooked needle on day 5 of pseudopregnancy under ether anaesthesia. Animals were housed in a controlled environment (22–24°C) and kept under controlled conditions  with free access to standard rat chow and water. The University of Illinois at Chicago animal care and use committee approved the animal care and handling.6 cells/ well). They were allowed to attach 3–4 hrs before washing in PBS to remove erythrocytes. Cells were incubated in RPMI 1640 without phenol red (Mediatech, Whashington DC), containing 1% CD-FBS (HyClone Laboratories Inc, Logan, UT), and treated with or without high levels (5 μM [2α (Sigma, St. Louis, MO) for 12 hr). After incubation, the cells were harvested in cold PBS, quick frozen in liquid nitrogen and kept at -80 C until RNA isolation.For each experiment 15 pseudopregnant rats were used. Decidual cells were isolated as previously described . Cells wls 5 μM ,11) of P of P6 ce2α or vehicle were pooled independently and subjected to cDNA array. cDNA probes were generated from 4 μg of total RNA in a reverse transcriptase reaction using a mix of dATP, dTTP and dGTP  plus [α32P]-dCTP (Amersham, MO), and a mixture of primers specific to each gene present in the array. All probes used had 5 to 10 × 106 cpm and the difference between control and experimental probes in each assay was less than 10%. cDNA were hybridized to an Atlas Rat 1.2 Array (#7854-1) nylon membrane (Clontech, CA). Hybridization and post-hybridization washes were performed according to the manufacturer's protocol. The signal was scanned with a phosphorImager (Molecular Dynamics, CA) after 48 h of exposure. Control and experimental RNA were always processed in parallel.Total RNA was isolated from primary decidual cells (PDC) by using a RNAII isolation kit , following the manufacture's instructions. RNA isolated from wells treated with either PGFSpot intensities from scanned membranes were analyzed using the AtlasImage 1.5 software (Clontech, CA). Grids were orientated manually and adjusted to ensure optimal spot recognition using AtlasImage's fine tuning tools, discarding spots with dust or locally high background. The software makes the analysis for background correction and normalization versus housekeeping genes. It also calculate the ratio and indicates which genes has a ratio > 2 (up regulated), or ratio < 0.5 (down regulated) or 0.5 < ratio < 2 . Gene expression data were normalized using the Sum method included in the Atlas Image software. Data points where the expression was not greater than two standard deviations were discarded. For the final analysis, data points were the averages from triplicates and any non-reproducible data were discarded. The relative RNA expression with differences between control and treatment being higher or equal to 2 and lower than 0.5 were considered significantly different .2α .From the total genes available in the rat 1.2 membrane arrays, only 20% of the genes were detected in rat primary decidual cells. Sixty genes were significantly down regulated, whereas only six genes were up regulated by PGF2α in primary decidual cells are genes involved in the regulation of the ECM. Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (uPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase – all of which were significantly up regulated by PGF2α (Table The majority of genes whose expression was affected by PGF2α. Only proteasome component C9 was up regulated after PGF2α treatment (Table Messenger RNA encoding proteasome Iota, proteasome component C2, proteasome subunit RC7-I, 26-S proteasome regulator and proteasome component C3 were down regulated by PGF2α treatment (Table 2α down regulated the expression of serum glucocorticoid kinase (SGK), casein kinase I, extracellular signal-regulated kinase (ERK-1), male germ cell-associated kinase (MAK), and Wee tyrosine kinase. PGF2α also significantly inhibited Crk-associated kinase (CAS), calcium calmodulin kinase II (CAMKII) and IV (CAMKIV), phospholipase C1, and protein phosphatases such as phosphatase 2A and protein tyrosine phosphatase 1B (Table The mRNAs encoding for 14-3-3ε and z/δ proteins, and Annexin IV were down regulated in PDC after PGF3), guanine nucleotide-binding protein α stimulating (GN-BP α stimul.), Rab 11A and guanine nucleotide-binding protein α12 subunit (GN-BP α12) gene expression were down regulated by PGF2α (Table 2α treatment as well (Table 2α. Conversely, PGF2α stimulated the expression of basic fibroblast growth receptor factor 1 (bFGRF1) (Table The 5-hydroxytryptamine receptor 2A (HTR2A), adenosine A2A and A2B receptors (ADORA2A and ADORA2B respectively), guanine nucleotide-binding protein G α3 subunit (GN-BPG α2α Table . The mRNll Table  and leuk1) Table .2α in the uterus need to be under tight control to avoid interference with the establishment and progression of pregnancy. Pathophysiological elevations in PGF2α lead to excessive uterine contractions. Therefore, PGF2α production must be avoided to maintain a quiescent uterus [2α can target the decidua, an endocrine tissue whose integrity is fundamental for the success of implantation and for the progression of pregnancy. Our results demonstrate that elevated PGF2α down regulate the expression of decidual genes related to the proteasome protein component system, those involved in trafficking and signal transduction and genes associated with G-proteins, growth factors, chemokines and cytokines. However, interestingly, the main effect of PGF2α is related to the turnover and degradation of the ECM which provides mechanical strength to the tissue. Thus, in addition to inducing uterine contractions, PGF2α can have a noxious impact on the success of implantation and progression of pregnancy, at least in part by deregulating the turnover of the ECM in the decidua.Levels of PGFt uterus . In this2α is directly or indirectly related with the turnover and degradation of the ECM. Genes such as gelatinase A (MMP-2), TIMP2 and 3 [The largest group of genes down regulated by PGFP2 and 3 ,15, cathP2 and 3 , carboxyP2 and 3 . TGF-β iP2 and 3 . TGF-β cP2 and 3 -21.Endothelin-1 is a peptide characterized as a potent endothelial cell-derived vasoconstrictor. It is synthesized as an inactive precursor (preproendothelin) and processed to a mature active form (endothelin) by zinc metalloproteinases. The active form, endothelin-1, promotes synthesis of collagen types I and II by fibroblasts, affects the ECM remodelling and promotes the proliferation of mesangial cells . Endothe2α. This protein belongs to a family of intracellular proteins that binds membrane phospholipids in a calcium-dependent manner. Thus, it can also inhibit phospholipase A2 (PLA2) [Annexin IV  was differentially down regulated by PGF2 (PLA2) . ANX4 ca2 (PLA2) . It has 2 (PLA2) ,25.1, h2 and acidic calponin, and can be identified by the individual C-terminal tail sequences. The c-terminal sequences regulate actin association and the cytoskeleton [Calponin (CaP) has three genetic isoforms, hskeleton ,27. It iskeleton . On the skeleton .1 antiproteinase, plasma proteinase-Iα inhibitor, basic fibroblast growth receptor factor 1, phosphorylase B and proteasome component C9 were the only genes up regulated by PGF2α in PDC. Pro-stromelysin is a 53 kDa peptide and a pro-MMP3 precursor and is directly related with the ECM regulation [2α was BMP4, a gene which appears to be involved in specific stages of embryo development and is [Pro-stromelysin, αgulation . Plasma gulation , an impogulation . Growth gulation . bFGRFs gulation -35. Anott and is .2α. Proteosomal component systems are the main non-lysosomal proteolytic structures of the cells that participate in the elimination of abnormal proteins, short half-life proteins, and proteins controlling cell cycle [2α.Proteasome component system proteins, such as proteasome Iota, proteasome component C2, proteasome subunit RC-7 I, 26 S-proteasome and proteasome component C3 were down regulated by PGFll cycle . During ll cycle . Kinase ll cycle . On the ll cycle . Decorinll cycle , which i2α and is another gene related to the ECM. Active CAS can modulate changes in cell motility and gene expression by the various MAP kinase cascades, and modify the organization of the actin cytoskeleton [2α was SGK, which is a transcriptionally-regulated serine/threonine protein kinase with 45–55% homology to the catalytic domain of Akt/PKB protein kinase A [Crk-associated substrate (CAS) was down-regulated by PGFskeleton . Anotherkinase A . Skg is kinase A . Trafickkinase A .2α. The first and most important point of control of PP2A is at the transcriptional level. The increase of phosphatase activity corresponded with a decrease in the phosphorylation of cellular proteins in anchorage-dependent cells, but much lesser regulated in anchorage-independent cells [Protein phosphatase type 2A (PP2A) was down regulated by PGFnt cells .It is well known that members of the proinflammatory cytokine family can induce MMP expression in numerous tissues . Also, cin vivo on day 12 of pseudopregnancy (when the decidua undergoes regression) with that of the PGF2α treatment in rat PDC in vitro (data not shown). Interestingly, we found a 49% coincidence in the genes that were down regulated in both experimental situations. This coincidence suggests a relationship between the physiological regression and reorganization of the decidual tissue that occurs on day 12 of pseudopregnancy, with the pattern of gene expression in rat PDC after PGF2α treatment. On the other hand, many of the genes whose expression was affected by PGF2α in the rat PDC, are expressed in the decidua during decidualization and implantation. This suggests a possible connection between the action of PGF2α and the physiology of the decidual tissue. Moreover, the ECM is an important component of the decidualization and implantation process. Any modification in its turnover or degradation could affect the formation of the decidua, blastocyst invasion, and the timing of decidual regression and reorganization. It is known that the expression of MMPs/TIMPs plays an important role in the control of implantation. If PGF2α silences or decreases the expression of genes related with the systems aforementioned, it also could affect tissue remodelling by directly modifying the proteases involved in this process, or indirectly by affecting the ECM turnover and degradation, important in the accumulation of a spongy mass of tissue around each embryo during decidualization [2α's effects on the expression of genes involved in ECM turnover are indirect. Alterations of genes involved in different signalling pathways such as ERK-1, CaMCKII, PLCδ1 and G-protein subunits may impact the expression of a great number of transcripts.We have also compared the pattern of gene expression in rat decidual tissue lization . Most pr2α have a severe impact on the expression of numerous genes associated with the turnover of the ECM in the rat decidua. Future investigation should corroborate the differentially regulated genes, at the level of the message, protein, and in some cases, such as for the metalloproteinases, at the level of activity of the proteins. These data will contribute to the design of future studies on a cluster of gene candidates as targets of PGF2α action in this endocrine tissue.In summary, our results show, for the first time, that pathophysiological concentrations of PGF1 = phospholipase C delta 1; BMP 4 = bone morphogenetic factor 4; ADPRF 5 = ADP ribosylation factor 5; bFGFRF1 = basic fibroblast growth factor receptor F1; HTR2A = 5-hydroxytryptamine receptor 2A; ADORA2A = adenosine receptor A2A; ADORA2B = adenosine receptor A2B; GN-BPG α3 = guanine nucleotide-binding protein G α3; GN-BP α stimul. = guanine nucleotide-binding protein α stimulating; GN-BPG α12 sub. = guanine nucleotide-binding protein α12 subunitTIMP2 = tissue inhibitor metalloproteinases 2; TIMP3 = tissue inhibitor metalloproteinases 3; PAI 1 = plasminogen activator inhibitor 1; uPA = urokinase type; tPA = tissue type palsminogen activator; TGFβ = transforming growth factor β; SGK = serum glucocorticoid kinase; ERK-1 = extracellular signal-regulated kinase-1; MAK = male germ cell-associated kinase; CAS = crk-associated kinase kinase; CamKII = calcium calmodulin kinase II; CamKIV = calcium calmodulin kinase IV; PLC δEC and SFG carried out the decidualization, and primary decidual cell culture. EC isolated the RNA isolation, performed the cDNA array assay, the analysis of the results, and drafted the manuscript. GG conceived the study and edited the manuscript. All authors read and approved the final manuscript.


It is the certain fate of all antibacterials to be fought off eventually by the pathogens they target. We need new ways to defeat disease, and we will need them forever Streptococcus pneumoniae strains in the United States will resist both penicillin and erythromycin, according to a recent prediction from the Harvard School of Public Health. The forecast, based on mathematical modeling, was published in the spring of 2003. It's too early to tell whether that prediction is precisely on track, according to the senior author on that paper, Marc Lipsitch. But no one doubts that multidrug resistance in this common bug—responsible for diseases that range from sinus trouble and ear infections to meningitis and pneumonia—is speeding up.By next summer, more than 40% of It is the certain fate of all antibacterials to be fought off eventually by the pathogens they target. The fact that the process is accelerating has been alarming public health officials for some time, especially in the United States. We need new ways to defeat disease, and we will need them forever.Staphylococcus, Streptococcus, and Enterococcus.Antibiotics have traditionally been plucked from nature's battleground. For billions of years, tiny organisms have engaged in an arms race, hurling toxic molecules at each other in the struggle to prosper. Nearly all of today's antibiotics are versions of weapons long wielded by microbes and fungi. Chemical synthesis of entirely human-created antibiotics has so far yielded only fluoroquinolones, a group of broad-spectrum antibiotics that includes Cipro, which became famously scarce during the 2001 anthrax scare, and linezolid (trade-named Zyvox), which is effective against some resistant strains of Escherichia coli from transcribing RNA.The usual way to find a new antibiotic has been laborious screening of immense libraries of compounds, natural and otherwise. Some argue that screening chemical libraries is approaching a deadend. There may be diminishing returns from screening, but it's not quite dead yet: in October, researchers at the University of Wisconsin at Madison reported a new class of bacterial RNA polymerase inhibitors with antibiotic potential. They were found by screening for molecules that prevent Christopher T. Walsh of Harvard Medical School says screening's problem may be simply that libraries aren't good enough. Marine organisms have not been studied well, he points out, and 90% of organisms in the biosphere can't be cultured in standard ways. He says, “We're missing 90% of them every time we go and look in nature.”Walsh is doing his bit to create new libraries. He and his colleagues have recently employed combinatorial biosynthesis to learn how to use part of the machinery for assembling cyclic peptide antibiotics to control their architecture. The result was a small library of natural product analogs, some of which have improved antibiotic activity against common bacterial pathogens. “There are dozens of such enzymatic domains that in principle one could clone, express, and test with other substrates. I view that as the kind of thing we should do,” he says. For example, Walsh suggests, it is a reasonable approach to second-generation improvement of daptomycin, the antibiotic most recently approved for sale in the United States.Walsh collaborates with Chaitan Khosla of Stanford University on finding ways to make existing antibiotics better. They are studying biosynthesis of rifamycin, an antibiotic that is increasingly less effective against its prime target, tuberculosis (TB) see . “In the“Basically, what we do is to try and figure out new ways to hijack the biosynthesis of antibiotics in nature so as to modify their structures with the goal of improving them,” Khosla explains. He works with an important class of natural antibiotics called polyketides that have generated dozens of drugs, including erythromycin.PLoS Biology). Among them are the anthracyclines, a group of anticancer drugs and antibacterials that includes tetracycline. In this issue of PloS Biology, Khosla and his colleagues report that they can make selective positional modifications in existing anthracycline antibiotics by starting in a different way with a different starting molecule. The molecule came from a natural anthracycline antibiotic, an estrogen receptor antagonist called R1128. R1128 is made via two modules of enzymes that work sequentially; the first module starts the process, and the second completes it. This division of labor permitted the researchers to tack the first R1128 module onto two other enzyme systems, thus engineering completely new anthracyclines. Some were more active in two types of assays than the natural parent molecule. “One setting was an assay on an estrogen-sensitive cancer cell line. Another setting was an assay to probe activity of an enzyme that's of particular interest in Type 2 diabetes, called glucose-6-phosphate translocase.” The work also revealed fundamental mechanistic features of the polyketide synthases, Khosla says.Polyketides are secondary metabolites (which give their producers a competitive advantage in their environment) produced mostly by bacteria and fungi and made by a complex and structurally diverse family of enzymes called polyketide synthases , the United States Department of Defense's (DOD) central research and development organization, and once the researchers realized they wanted to develop drugs against three agents that have been considered bioterrorism threats — The most radical approach to new antibiotics may be the resurrection of an old idea: bacteriophage therapy see . Late inPhage were the model organisms of choice for genetics research in the 1930s and 1940s, but became less fashionable as research tools when investigators moved on to eukaryotes. A few held on, like Ry Young of Texas A&M University, who has made phage-induced cell lysis his life's work. “The cell is basically genetically dead as soon as the phage goes in there, but it will keep living as sort of an infected zombie for as long as the phage wants it to, with virus particles accumulating inside the cell,” he explains. “Only when the phage is ready and has decided that it's the right time will it pull the trigger. And the cell blows up.” The freed phage then spew forth to infect new cells.Antibiotic resistance has led to new interest in phage therapy by several small biotech companies. Young continues basic research at Texas A&M, but has also joined one of them, GangaGen, providing bacteriophage expertise to its labs.Phage do kill pathogenic bacteria effectively, and they do it without penetrating human cells, which they can't even recognize. So what is keeping phage therapy out of the clinic? Problems that some doubt can be overcome.Because bacteria develop resistance to phage rapidly, phage therapy companies will need to direct cocktails against a single pathogen, according to Vincent Fischetti at The Rockefeller University. Phage are also antigenic, and the antibodies they stimulate will neutralize their effects during subsequent treatment, he says. But the chief problem appears to be regulatory—regulatory in the political, rather than the genetic, sense. When bacteriophage package their DNA, they occasionally include varying amounts of their hosts' DNA, too. This miscellany, Fischetti points out, is likely to make the Food and Drug Administration unhappy. “Phage normally are very fragile, their tails break, so lot-to-lot homogeneity could be a problem too,” he adds. “So even though it will work, I think they'll have an uphill battle.” Phage may well enter agricultural or veterinary use, he predicts, but are probably not going to be available to patients in the United States any time soon.Strep pyogenes, pneumococci, Strep pneumoniae, Bacillus anthracis, Enterococcus faecalis, and group B Strep. The beauty of these enzymes is that they are targeted killing. You only kill the organism you intend to kill, without destroying or affecting the surrounding organisms that are necessary for health.”Fischetti chose a different approach to phage therapy. It does not rely on phage themselves, but on enzymes that phage produce to smash their way out of their host bacteria so they can infect new hosts. He and his colleagues employ these enzymes externally to kill bacteria. He reports, “We now have enzymes that will kill Pneumococcus, Staphylococcus, and group A Strep on contact with mucous membranes. The strategy might prevent bacterial infections from spreading in close quarters like hospitals, nursing homes, and daycare centers. Fischetti says, “Clinical trials would tell us how often we had to treat, but more important, we'd have a reagent that could treat people who walk out the door of the hospital to eliminate or reduce the transmission of resistant organisms into the community. We don't have that capability right now.”The enzymes can be loaded into a nasal spray that wipes out pathogens such as Bacillus anthracis spores, preventing them from germinating and seething through the bloodstream, producing deadly toxins. An IV drip would be started after exposure to the spores. The method, Fischetti reports, is already successful in mice; clinical trials will determine how long treatment must be continued, perhaps a week or so. They have also eliminated septicemia from pneumocci with the same intravenous method.Fischetti and his colleagues have moved on to using the enzymes systemically to wipe out Up to now the enzymes must make contact with bacteria to kill, but Fischetti is hoping that a new generation of engineered enzymes will be able to kill pathogens inside cells too. A second disadvantage is that they are effective only against gram-positive bacteria, although that group includes many vicious pathogens.But phage enzymes seem to offer one very big advantage: resistance to them has yet to develop. Fischetti says, “We've tried very hard to identify resistant bacteria, but so far we haven't found resistant organisms in all three of the enzymes we're working with. It appears to be a very rare event, much rarer than resistance to antibiotics.” Fischetti cautions against expecting that gladsome state to last forever, but he points out that even if widespread resistance takes the same 40 or 50 years that antibiotics required to become significantly resistant, phage enzymes could buy researchers decades for inventing other approaches.There is no shortage of ideas for unearthing new antibiotic candidates. Why are they so slow to enter medical practice? The bottleneck, researchers agree, lies in the development process of turning them into effective therapies. Several researchers blame the big pharmaceutical companies that got so big by leading the way to new drugs for battling infectious disease, but in recent years have dropped out. Fischetti complains, “These are the big companies that have the money to develop antiinfectives, but they leave it to small biotech companies, and it's not going to happen as rapidly as it should. I think it's really unconscionable for these big companies to drop the ball because it's not going to be a billion-dollar market for them and that's what they're looking for.”Half a billion at least, says Francis Tally, a big pharmaceuticals veteran who is now chief scientific officer at Cubist Pharmaceuticals, a biotech company located in Lexington, Massachusetts. According to Tally, Cubist produced daptomycin, approved in September 2003, by licensing it from Eli Lilly, which shelved the new compound after concluding its potential market was only $250 million.But, Tally argues, the size of the market is not the only barrier to new antibiotics. Combinatorial chemistry and the genomics revolution have simply not delivered on their early promise. “The pipeline is very dry,” he says. “There's been a real lag at the basic research level.”“Antibiotic discovery is hard,” Shapiro says. “It's a huge long process to get a decent antibiotic.” Walsh agrees. “It's easier to find inhibitors of particular enzymes for particular processes—and a very long road to convert that into something for development.”In the meantime, there is a rising clamor to slow down the rate at which bacteria develop resistance. Doctors are exhorted to cut back on prescribing antibiotics and decline to prescribe for viral diseases, which antibiotics can't combat, even when their patients badger them.But even if antibiotic consumption slowed, we will still need new antibiotics. “I always say it's not a matter of if, it's only a matter of when,” says Walsh. “There will always be a need for new antibiotics because the clock starts ticking on the useful lifetime of any antibiotic once you start to use it. That cannot be argued.”


Ethanol inhibits proliferation in astrocytes, an effect that was recently linked to the suppression of phosphatidic acid (PA) formation by phospholipase D (PLD). The present study investigates ethanol's effect on the induction of apoptosis in astrocytes and the formation of ceramide, an apoptotic signal. Evidence is presented that the formation of PA and ceramide may be reciprocally linked during ethanol exposure.3H]-serine, ethanol caused a dose-dependent, biphasic increase of the [3H]-ceramide/ [3H]-sphingomyelin ratio after 1 and 18 hours of incubation. As primary alcohols such as ethanol and 1-butanol were shown to inhibit the phospholipase D (PLD)-mediated formation of PA, a mitogenic lipid messenger, we tested their effects on ceramide formation. In astrocytes prelabeled with [3H]-serine, ethanol and 1-butanol, in contrast to t-butanol, significantly increased the formation of [3H]-ceramide. Moreover, exogenous PA, added to transiently permeabilized astrocytes, suppressed ethanol-induced [3H]-ceramide formation. Vice versa, addition of C2-ceramide to astrocytes inhibited PLD activity induced by serum or phorbol ester.In cultured rat cortical astrocytes, ethanol (0.3–1 %, v/v) induced nuclear fragmentation and DNA laddering indicative of apoptosis. Concomitantly, in cells prelabeled with [We propose that the formation of ceramide in ethanol-exposed astrocytes is secondary to the disruption of phospholipase D signaling. Ethanol reduces the PA:ceramide ratio in fetal astrocytes, a mechanism which likely participates in ethanol-induced glial apoptosis during brain development. The proliferation of astrocytes is stimulated by polypeptide growth factors such as PDGF, EGF, bFGF and IGF-1 acting on cellular signaling pathways which involve tyrosine kinases, protein kinase C, and the Ras-Raf-MAP kinase pathway . AstroglThe molecular target of ethanol's antimitogenic actions in astroyctes is not known with certainty, but inhibitory interactions of ethanol with lipid signaling pathways have been implicated . Our groIn our previous work, we have found that astroglial PLD is activated by mitogenic factors including fetal calf serum (FCS), PDGF, and phorbol ester, and we observed that ethanol reduced both astroglial proliferation and PA formation in a parallel manner. 1-butanol reduced PA formation and DNA synthesis with the same potency while t-butanol was inactive for both effects . More relyso-PA, a potent mitogen in many cell types [Recent findings from other groups are also compatible with a central role for the PLD signaling pathway in ethanol toxicity in astrocytes. First, several mitogenic factors including those that are known to be particularly sensitive to ethanol activate PLD activity in astrocytes. This holds true for PDGF , acetylcll types ,26. Takede novo-synthesis, inhibition of ceramide breakdown, or activation of  sphingomyelinase (SMase), an enzyme which catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine . In . In de nine (see ,34. For 2-ceramide (50 μM) but not with t-butanol (1%, v/v) (not illustrated). In parallel experiments, ethanol caused fragmentation of nuclear DNA in serum-starved astrocytes which is reflected by "DNA laddering" on agarose electrophoresis  mimicked the effect of ethanol. Ethanol at 0.3% (v/v) was almost as effective as 1 % , staining of the cells with Hoechst 33258, a dye intercalating into DNA, revealed condensation and fragmentation of the nuclei which was visible after 16 hrs; the maximum effect was observed after 21 hours Fig. . Higher 3H]-ceramide was measured after labeling sphingomyelin with [3H]serine. Under basal conditions, the ratio of [3H]-ceramide to [3H]-sphingomyelin ("C/S ratio") was approximately 1:30. This ratio was not significantly changed during serum withdrawal  but did not significantly change the total labeling of the large pool of [3H]-sphingomyelin by [3H]-serine (data not shown). As the total incorporation of [3H]-serine into [3H]-sphingomyelin was somewhat variable between individual preparations, we used the C/S ratio to calculate ethanol-induced changes. As shown in Figs. Formation of H-ceramidal Figs. . The incThe rapid and transient phase of ceramide formation occurred within 15 min and reached a maximum at 1 hour after addition of ethanol . A second increase gradually developed after 4 hours and reached a maximum at 18 hours of ethanol exposure . At this later time point, staurosporine (1 μM) caused an increase of the C/S ratio to 7.97 ± 1.78 % .To investigate whether ceramide formation is secondary to a disruption of PLD signaling, we used the isomeric alcohols, 1-butanol and t-butanol. 1-Butanol – but not t-butanol – is a substrate of PLD for transphosphatidylation and leads to the formation of phosphatidyl-1-butanol at the expense of PA. The correlations between butanol exposures and disruption of PLD signaling (i.e., suppression of PA formation) in astrocytes were documented in detail in our previous work . In the 3H]-ceramide levels (data not shown). Basal ceramide levels were also unchanged if exogenous PA was added to the astrocytes in the absence (data not shown) or presence of SL-O  of streptolysin-O (SL-O) to introduce the membrane-impermeable PA into the astroglial cytosol . In prelL-O Fig. . HoweverL-O Fig. . At the L-O Fig. . At the L-O Fig. .2-ceramide (50 μM) slightly but non-significantly reduced basal PLD activity by 30%  84 and 93 % and (for PDB stimulation) 53 and 64 %, respectively.As the previous experiments indicated an inhibitory effect of the PLD pathway on ceramide formation, the following experiment tested a possible effect of ceramide on PLD activity measured by the transphosphatidylation assay. In these experiments, the membrane-permeable C30% Fig. . However30% Fig.  or by PD30% Fig. , C2-ceraWe have previously documented that ethanol suppresses signaling through the mitogenic phospholipase D (PLD) pathway, and we and others have provided evidence that this effect may be responsible for the antiproliferative actions of ethanol in astrocytes ,17,23. T2-ceramide , and sphingomyelinase activity was actually shown to be responsible for ethanol-induced ceramide formation in a recent study [de novo-synthesis of ceramide or inhibition of ceramidase.It should be noted that the present experiments do not unequivocally identify the mechanism of ceramide formation. We used [nt study . HoweverThe important findings of this study relate to the interaction between lipid second messenger pathways. It was known from previous work (see Introduction) that PA, the product of phospholipase D (PLD), mediates mitogenic stimulation in astrocytes whereas formation of ceramide by sphingomyelinase activation accompanies apoptosis. We now tested the hypothesis that ethanol causes astroglial apoptosis by inhibiting PLD and, as a consequence, stimulates the sphingomyelinase pathway. The results shown in Figs. 2-ceramide inhibits PLD activity at a concentration as low as 10 μM. Basal PLD activity was only slightly inhibited, but the increases of PLD activity induced by addition of serum or phorbol ester were strongly antagonized. This finding in astrocytes corroborates previous reports that ceramide can inhibit PLD signaling in peripheral cell types [We also probed the reciprocal effects of ceramide signaling on PLD activity. The results shown in Fig. ll types ,39. It rll types . Interesll types ,16. At tll types . The molll types ,43.In summary, the present results give evidence of a cross-talk between lipid-signaling pathways in astrocytes such that the product of PLD, namely PA, inhibits ceramide formation whereas ceramide inhibits PLD activation Fig. . The exp3H]-Serine and [3H]-glycerol were from Biotrend (Köln, Germany). Ceramide (from bovine brain), C2-ceramide (N-acetyl-D-erythro-sphingosine), and staurosporine were from Alexis (Lausen, Switzerland). Hoechst 33258, L-α-phosphatidic acid  and 4β-phorbol-12β,13α-dibutyrate (PDB) were from Sigma (Deisenhofen, Germany); most other chemicals and TLC plates were obtained from Merck (Darmstadt, Germany) or Roth (Karlsruhe, Germany) at the highest purity available. Fetal calf serum (South America) was from Invitrogen, cell culture materials were from Sarstedt (Nürnbrecht, Germany). Phosphatidylethanol (PEth) standard was synthesized as described [[escribed . Recombiescribed .2). The growth medium was DMEM containing 10% fetal calf serum (FCS), 2 g/l NaHCO3, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37°C in a 95/5% mixture of air and carbon dioxide. For the experiments, astrocytes were grown for two weeks in culture and were used when they reached confluency. As judged by GFAP immunostaining, these cultures contained >90% astrocytes.Astrocyte-rich cultures were prepared from newborn rat pups. Cerebral hemispheres were collected, meninges and blood vessels were removed, the brain tissue was dissociated by trituration, passed through a 50 μm nylon mesh, and the cells were seeded onto plastic culture dishes (14,000 cells per cm2-ceramide 50 μM or staurosporine 1 μM) for 24 hrs in serum-free medium. Subsequently, cells were washed and fixed with ice-cold methanol/acetic acid (3:1). Dried and re-hydrated cells were stained with bisbenzimide (Hoechst 33258, 1 μg/ml) solution for 10 min, washed and sealed in gelatin. Photographs were obatined using a Leica Leitz DMRB fluorescence microscope and a Nikon Digital Camera DXM.Cells were seeded on microplates (5,000 cells per 200 μl) and incubated with different apoptogens (ethanol 0.3 and 1 %, CThe test was carried out as described  with som3H]-serine (1 μCi/ml) to astrocytes kept in growth medium (DMEM plus FCS) for 72 h. After washing, cells were incubated in serum-free medium with ethanol, butanol, C2-ceramide or staurosporine (see Results). C2-ceramide or staurosporine were added in DMSO; the final DMSO concentration was < 0.1 %. At the end of the incubation, cells were fixed with methanol, transferred and extracted first with with chloroform: methanol (1:1), then with chloroform: methanol: water (10:10:9) to separate water and lipid phases. After addition of ceramide and sphingomyelin standards, the lower (lipid) phase was evaporated, taken up in chloroform:methanol (3:2) and separated by thin-layer chromatography (TLC). For the determination of [3H]-ceramide, we used one-dimensional TLC . For the determination of [3H]-sphingomyelin, we used two-dimensional TLC (TLC plates Merck 1.05721); solvent I was chloroform/methanol/25% aqueous ammonia (13:7:1), solvent II was chloroform/ methanol/water/acetic acid (30:30:2:5). 3H-lipids were visualized by using iodine vapor, spots were scraped and suspended in scintillation cocktail (Lumasafe Plus), and radioactivity was measured by liquid scintillation counting (Packard 1600 CA). Formation of [3H]-ceramide was calculated as percentage of radioactivity found in [3H]-sphingomyelin.Phospholipids were labeled by addition of [2PO4, 60 mg Na2HPO4 × 2 H2O, 100 mg glucose in 1 L of sterile water). PA  was added as a suspension in buffer prepared by sonication. After 15 min, the cells were washed and incubated in serum-free, calcium-containing DMEM. Formation of [3H]-ceramide was determined as described above. Ethanol, if present, was present throughout the incubation period. Using 144 ng/ml of streptolysin-O, this procedure yielded transient permeabilization of > 80% of astrocytes followed by repair of the pore in > 80% of permeabilized cells. The procedure was previously found to allow the entry of approx. 106 molecules of PA per cell [For the introduction of PA, astrocytes were transiently permeabilized with streptolysin-O . Brieflyper cell .3H]-glycerol (1 μCi/ml) for 24 h in order to label phospholipids. More than 60% of the phospholipid label was associated with phosphatidylcholine (not illustrated). Subsequently, the cells were washed and re-exposed to medium containing ethanol (2 %) and PDB (1.0 μM) or FCS (10 %, v/v) as stimulators. PDB and C2-ceramide, when used, were dissolved in DMSO (end concentration of DMSO < 0.1 %). After 30 min of reaction time, the cells were washed in cold phosphate-buffered saline and extracted as described above for ceramide determinations. After addition of phosphatidylethanol (PEth) and PA standards, aliquots of the lipid phase were separated by two-dimensional TLC (TLC plate Merck 1.05721) using chloroform/methanol/25 % aqueous ammonia (13:7:1) for the first run and the upper phase of ethylacetate/isooctane/ acetic acid/water (13:2:3:10) for the second run. Individual phospholipids were stained by iodine, and the spots corresponding to PEth, PA and phosphatidylcholine (PC) were isolated and counted for radioactivity in a scintillation counter. To determine PLD activity, formation of [3H]- PEth was calculated as percentage of [3H]-PC.Phospholipase D activity was determined using the transphosphatidylation assay . For thiData are shown as means ± SEM of N experiments whereby N refers to the number of different astroglial preparations from different animals. Results were obtained from two replicate dishes which were pooled to represent a single experiment. Statistical calculations were performed by GraphPad InStat 3.0 program package, using analysis of variance (ANOVA) of paired or unpaired data as indicated in text and figure legends.Cer, ceramide; FAS, fetal alcohol syndrome; FCS, fetal calf serum; PA, phosphatidic acid; PC, phosphatidylcholine; PDB, 4β-phorbol-12β,13α-dibutyrate; PEth, phosphatidylethanol; PKC, protein kinase C; PLD, phospholipase D; SL-O, streptolysin-O; SM, sphingomyelin.B.S. carried out the cell culture experiments and phospholipid measurements and participated in the experiments concerning apoptotic markers. S.J. contributed substantially to the apoptotic marker experiments. K.L. participated in the design of the study and the final draft of the manuscript. J.K. conceived and supervised the study, performed the statistical analyses and drafted the manuscript.


Medical education is increasingly being conducted in community-based teaching sites at diverse locations, making it difficult to provide a consistent curriculum. We conducted a randomized trial to assess whether students who viewed digital lectures would perform as well on a measure of cognitive knowledge as students who viewed live lectures. Students' perceptions of the digital lecture format and their opinion as whether a digital lecture format could serve as an adequate replacement for live lectures was also assessed.Students were randomized to either attend a lecture series at our main campus or view digital versions of the same lectures at community-based teaching sites. Both groups completed the same examination based on the lectures, and the group viewing the digital lectures completed a feedback form on the digital format.There were no differences in performance as measured by means or average rank. Despite technical problems, the students who viewed the digital lectures overwhelmingly felt the digital lectures could replace live lectures.This study provides preliminary evidence digital lectures can be a viable alternative to live lectures as a means of delivering didactic presentations in a community-based setting. Medical education is increasingly being conducted in community-based teaching sites outside of the traditional academic medical setting , At the Our institution has a community integrated structure where medical students spend the clinical portion of their training in one of six community campuses spread throughout the State of Michigan. Although this structure has many advantages, it is difficult to provide a consistent educational experience for the students. To help address this challenge, we implemented an all-day lecture series held at one of the community campuses two weeks before the end of the internal medicine clerkship.The students and faculty presenters from other campuses traveled from their home campus to the campus hosting the lecture series. End-of-clerkship feedback from the students has indicated the lecture series is both valuable and well received. Traveling to the host community, however, was inconvenient and time consuming for both students and faculty presenters. In addition, it is not practical for students at our rural medicine campus to attend due to the distance (approximately 400 miles) from the other communities.There is evidence that delivering the audio from a lecture in combination with the presenter's slides can be an effective means of delivering lectures at remote sites, and may even be as effective as traditional lectures,4. We saStudents taking the third-year required internal medicine clerkship during the 2003–2004 academic year at our institution were offered the opportunity to participate in the study. Those agreeing to participate were randomized into to one of two arms of the study. The random assignment of students to the two arms of the study was done within each community to control for the potential of community differences. The control group traveled to the host community campus and attended the live lectures with their colleagues who chose not to participate in the study. The experimental group stayed at their home campus on the same day and completed a parallel set of CD-ROM-based multimedia modules made from digital recordings of the previous year's lectures. They completed these digital lectures in computer laboratories in either the community campus office or within one of the teaching hospitals.The series included six lectures covering asthma, coronary artery disease (CAD), acute renal failure, liver disease, thyroid disease, and antibiotic pharmacology. The Clerkship Education Committee chose these topics based on their perceived importance and consistency with the Society for General Internal Medicine/Clerkship Directors of Internal Medicine (SGIM/CDIM) Curriculum Guide.Between the 2002–2003 academic year when the lectures were taped and the 2003–2004 academic year when the study was conducted, the clerkship faculty decided to revise the lecture series to be more case-based though the topics were kept the same. Two of the lectures, CAD and renal failure, were not modified and kept as consistent as possible with the previous year in order to conduct the study. Though there might have been minor inconsistencies between the digital and live versions of these two lectures, the same faculty member presented each lecture during both the 2002–2003 academic year when the lectures were taped and the 2003–2004 academic year when presented live. The two lecturers also attempted to keep the live lectures as consistent as possible with the digital lectures and used the same slides that they had used the previous year. While the format of the other four lectures changed, the material covered and instructional objectives remained consistent.At the end of the live lecture series, students were asked to complete a short examination that included four to five questions based on each of the six lectures. These questions were written by the presenters of the lectures and designed to assess student mastery of the lectures' key objectives. The students were informed that the purpose of the examination was to provide them with feedback on the mastery of the material and the presenters with feedback on the effectiveness of the lectures, and would not impact on their clerkship grade. After the students completed the exam, they were given a copy that included the correct answers and a short explanation for the correct answer. The exam forms contained no student identifiers, but students in the control group were asked to indicate on the examination form that they had agreed to participate in the study so they could be differentiated from the students who had chosen not to participate in the study.Students in the experimental arm of the study completed the same examination in their home community after they had completed the digital lectures. They were also asked to complete a short feedback form asking whether they had any technical problems using the modules, to rate their agreement with what the researchers felt to be three potential advantages and three potential disadvantages of the modules, and whether they felt the modules could serve as a suitable replacement for live lectures.The specific questions are listed below.Advantages of the Modules• Convenience of viewing the presentations when you choose.• Avoiding having to travel to another community for an all day lecture series.• Ability to keep copies of these presentations for use in the future.Disadvantages of these modules• Inability to ask questions of the presenter• Lack of group interaction/discussion of a topic• Just not like being in the room with the presenter. They included digitized video and audio from the taped presentation inserted as a window in the PowerPoint® slides from the presentation. As students displayed each of the slides, they were able to observe the presenter in the multimedia window discussing the slide that was being viewed.The CD-ROM modules were created using a technique developed by the first author. A manual outlining how to develop these modules is available from Nine of the items on the exam focused on the material in the CAD and acute renal failure lectures, where the lecturers presented the lectures in the same format as they had used in the previous year when the lectures were taped. The remaining 20 examination items covered material in the other four lectures. Group differences were tested for statistical significance by both an independent sample t-test for means and a Mann-Whitney test for ranks. A Levene's test for equality of variance between the two groups was also performed. These analyses were conducted separately for the subset of items covering the material in the CAD/acute renal failure lectures and the other four lectures. A power analysis was conducted to assess the magnitude of the difference between the groups that would likely be detectable given the number of students participating in the study. The coefficient alpha reliability of the exam was also estimated. The data were presented descriptively using means, standard deviations and mean ranks within the control and experimental groups. All analyses were conducted using the Statistical Package for the Social Sciences version 11. Approval for the project was obtained from the University Committee for Research Involving Human Subjects within our institution.A total of 96 students completed the internal medicine clerkship during the 2003–2004 academic year. As described below 56 of the students were eligible to participate in the study. A total of 29 students or 52% of the eligible students agreed to participate in the study. Complete data were available for 12 students who attended the live lectures and 17 students who completed the digital lectures.During the first rotation, there were some technical problems in a demonstration of the digital lectures. The net result was that very few students chose to participate during that rotation. During the second and third rotations, approximately two-thirds of the students agreed to participate. There were also 20 students who were ineligible to participate. These included six students from the rural medicine program at Marquette who do not participate in the live lecture series due to the distance from the other communities. Additionally, some of the communities conducted a fourth rotation of the internal medicine clerkship due to space limitations in the three regular rotations and a live version of the lecture series was not given for the students in the fourth rotation. These 20 students all completed the CD-ROM modules but because they could not be randomized between the live and digital formats, they were not able to participate in the study.Differences in the sample sizes for the two groups were due to some of the students in the live lecture group failing to mark that they were participating the study. During the second clerkship rotation, the proctor inadvertently failed to remind the students participating in the study to mark this information on their examinations when the exams were handed out.A power analysis indicated that with the number of subjects in the study, it would be possible to detect differences of nine tenths of a standard deviation with a power of 80%, p < 0.05 for a one-tailed t-test.Table The Levene's test for equality of variance between the control and experimental groups was statistically significant (p = 0.03) for the CAD and renal failure items. The variation among the scores of students who observed the CD-ROMs was almost twice as large as for students who observed the live lectures. There was no statistically significant difference among the groups for the variance of the items from the other four lectures.The coefficient alpha reliability for the items covering CAD/renal failure and the items covering the other four lectures were 0.33 and 0.66 for the 9 and 20 item scales respectively and was 0.70 for the combined 29-item exam.The 17 students who completed the digital lectures also completed a short feedback form on their experiences and impressions of the digital lecture format. These data are presented in Table There were no statistically significant differences found between students who viewed the live and CD-ROM based lectures. The observed mean scores in the two groups were in fact almost identical. Unfortunately, the small sample size limits the power of the study and confidence in which we can assert that digital lectures can be as effective as live lectures in increasing students' knowledge. The study does suggest that it is unlikely that there are large differences in the performance of students who view CD-ROM based lectures as opposed to live lectures and adds to the growing body of literature concerning the effectiveness of technology for implementing distance learning in medical education.There was a statistically significant difference in variances among the two groups for the items covering the CAD/renal failure lectures. The standard deviation in the scores was twice as large for the students who completed the digital modules. The differences in the dispersion are also evident in the range of values in each group. It is not clear why there was more variation in the scores among the students who completed the digital modules. It may be related to the technical problems encountered by many of the students in accessing the modules, though one would expect this would have resulting in extending the lower tail of the distribution but not the upper tail. It may have also in part reflected the impact of discussions that occurred during the live lectures that may have reduced the variability among the students in their responses to the examination.Despite the fact that almost all of the students experienced some technical difficulties using the modules, they all agreed and most strongly agreed the modules could serve as an adequate replacement for live lectures. They were particularly appreciative of not having to travel to another community to attend didactic presentations and having the flexibility of viewing the modules at their convenience. Of the three potential disadvantages of the format that were listed on the feedback form, they felt their inability to ask a question of the presenter was the most important. In the future we are considering using a web-based bulletin board system as a means of allowing students to ask questions of the presenter.The number of students with technical difficulties viewing the modules was surprising. We had tested the modules on a variety of different computers with very few problems. In a few cases, the CD-ROMs we distributed apparently had not been copied correctly. Additionally, we switched the video formats from MPEG to Windows media files. We assumed there would be less compatibility problems with the Windows media files given that this is a format developed by Microsoft. Unfortunately, we later found out the Windows media files require software that was not shipped with earlier versions of Windows. We expect this was a significant cause of the technical problems the students experienced.We are now using a commercial software package which greatly simplifies the process of creating the digital lectures and allows them to be distributed over the Web as well as via CD-ROM requires no special software minimizing the compatibility issues. Students who completed the fourth rotation of the clerkship and did not participate in the study were provided with the new version of the modules. Only one of the 12 students indicated they had technical problems accessing the lectures off the CD-ROM disks on which the modules were distributed and that student was able to access the modules via the Web. Such combined Web and CD-ROM distance learning formats have been shown to be effective in a number of educational settings ,7.There were several important limitations in the study. First is the very small sample size which limited the power of the study for detecting differences in the performance of the students completing the live and CD-ROM based lectures. It also increased the likelihood the two groups of students were not equivalent. Since there were no student identifiers on the exams, it was not possible to compare the characteristics of the two groups. The outcome measure was a locally developed test. While the items were written by the presenters and based on the major objectives in their lectures, there was no assessment of validity other than content validity. It is also not clear the extent the findings of this study can be generalized to other digital lecture formats.Although the data collected in this study were limited, it provides some evidence that digital lectures are both well received by students and can provide a satisfactory substitute for live lectures from a performance standpoint.The author(s) declare that they have no competing interests.DJS designed the study, developed the feedback instrument on for the digital modules conducted the statistical analyses and wrote the first draft of the paper. GSF, HSF and KK developed and gave presentations, wrote questions for the knowledge examination and edited and helped revise the manuscript.The pre-publication history for this paper can be accessed here:


Releases of hazardous materials can cause substantial morbidity and mortality. To reduce and prevent the public health consequences (victims or evacuations) from uncontrolled or illegally released hazardous substances, a more comprehensive analysis is needed to determine risk factors for hazardous materials incidents.Hazardous Substances Emergency Events Surveillance (HSEES) data from 1996 through 2001 were analyzed using bivariate and multiple logistic regression. Fixed-facility and transportation-related events were analyzed separately.For fixed-facility events, 2,327 (8%) resulted in at least one victim and 2,844 (10%) involved ordered evacuations. For transportation-related events, 759 (8%) resulted in at least one victim, and 405 (4%) caused evacuation orders. Fire and/or explosion were the strongest risk factors for events involving either victims or evacuations. Stratified analysis of fixed-facility events involving victims showed a strong association for acid releases in the agriculture, forestry, and fisheries industry. Chlorine releases in fixed-facility events resulted in victims and evacuations in more industry categories than any other substance.Outreach efforts should focus on preventing and preparing for fires and explosions, acid releases in the agricultural industry, and chlorine releases in fixed facilities. Recent high-profile hazardous materials incidents, such as an explosion at a pharmaceutical supply plant in Kinston, North Carolina  and an eA more comprehensive analysis is needed to determine which risk factors are associated with releases of hazardous substances that result in victims or evacuations. The Hazardous Substances Emergency Events Surveillance (HSEES) system, maintained by the Agency for Toxic Substances and Disease Registry (ATSDR), is an active, multistate surveillance system designed to collect and analyze information about acute releases of hazardous substances. Data elements captured in HSEES include time, date, and day of the week when the event occurred; event type (fixed-facility or transportation-related event); geographic information; factors contributing to the release; type of chemicals released; information about injured persons (victims); industry responsible for the event; and information about decontaminations and orders to evacuate. Data from HSEES were analyzed to identify potential risk factors associated with chemical releases involving victims or evacuations during 1996–2001.HSEES events are defined as sudden, uncontrolled, or illegal releases of at least one hazardous substance that had to be removed, cleaned up, or neutralized according to federal, state, or local law. A substance is considered hazardous if it might reasonably be expected to cause adverse human health outcomes. Threatened releases are also included in HSEES, if the amount threatened to be released would have required removal, cleanup, or neutralization under federal, state, or local law and the threat led to an action to protect the public health (e.g., rerouting traffic, closing a road, or ordering an evacuation). Events involving only petroleum are excluded from HSEES.The purpose of HSEES is to reduce morbidity and mortality associated with acute releases of hazardous substances. The goals of the system are to: 1) describe the distribution and characteristics of hazardous substances emergencies, 2) describe morbidity and mortality of employees, emergency responders, and the general public resulting from hazardous substance releases, 3) identify risk factors for morbidity and mortality, and 4) identify strategies that might reduce future morbidity and mortality resulting from the release of hazardous substances.The pilot phase of the surveillance system was from January 1, 1990 through December 31, 1992. Data for 1996–2001, the most recent period for which complete data are available, were analyzed. Thirteen states participated in HSEES during the entire period analyzed: Alabama, Colorado, Iowa, Minnesota, Missouri, Mississippi, New York, North Carolina, Oregon, Rhode Island, Texas, Washington, and Wisconsin. An additional four states participated during portions of that period: Louisiana (2001), New Hampshire (1996), New Jersey (2000–2001), and Utah (2000–2001).State health department personnel used a variety of sources  to collect information about the hazardous events. Before January 1, 2000, data were entered into a computerized data-entry system designed by ATSDR and were transmitted quarterly to ATSDR for quality control checks and analysis. Beginning on January 1, 2000, data were entered into a web-based application that enabled ATSDR to instantly access the data.A standardized data-collection instrument was used to obtain information on each event. ATSDR provided the states with a training manual to ensure uniformity. A victim is defined as a person experiencing at least one documented adverse health effect  that probably resulted from the event and occurred within 24 hours after the release. The HSEES system does not identify the immediate cause of the adverse health effect other than the event itself. Official evacuations were recorded in the system if they were ordered by on-scene coordinators such as a fire or police chief, a member of a HazMat team, or some other type of official. Reasons for the evacuation are not captured.Industry codes for the type of industry responsible for each HSEES event were assigned according to the 1990 Industrial Classification System of the United States Census Bureau . The indData were analyzed using SAS  logisticFor each chemical reported, up to two entries for type of release could be selected from six choices (spill, air, fire, explosion, threatened release, other). To describe an event, release type was assigned in a hierarchic arrangement (fire and explosion, explosion, fire, air, spill, or threatened) according to all release types reported for an event. When an event had multiple release types, the highest rank was used to assign the release type. For example, an event that had a chemical released as both an air emission and a fire  would be classified as fire. Threatened releases were analyzed only for events with evacuations. The area of evacuation recorded if the evacuation zone was downwind/downstream of the release, a circular area, the building(s) or affected part of the building(s), both a circular area and downwind/downstream, or if no criteria were used.For fixed-facility events, industry category and factor contributing to the release  were also included. Although up to two factors could have been selected for each event, we analyzed only the first factor selected, because only 13% of events had a second factor selected and specific combinations of factors were too sparse to provide meaningful analysis.We examined the likelihood of an event resulting in a victim or evacuation for specific substance and industry categories separately. Substance categories were coded as yes/no; the referent group was all other substances. Similarly, industry categories were coded as yes/no; the referent group was all other industries. Because most transportation-related events were coded into the transportation industry (79%), the association between industry type and an event with a victim (or evacuation) was examined only for fixed-facility events. Mode of transportation, categorized as ground (i.e., truck), rail, water, air, and other, was also included in the analysis for transportation-related events.To further specify risk factors, multivariate and stratified analyses were performed to examine whether certain combinations of variables significant as univariate predictors were associated with a greater likelihood of an event with a victim or evacuation. A multiple logistic regression model was constructed to examine the potential interactions among time of day, weekday, and season. For fixed-facility events, analysis stratified by industry category examined the association between specific substances and an event with a victim or evacuation within industries.During 1996–2001, participating state health departments reported 39,766 events to the HSEES system. The analysis of risk factors for events with at least one victim excluded 568 events that involved only a threatened release, and thus focused on 39,198 events (29,974 (76%) fixed-facility events and 9,224 (24%) transportation-related events). At least one victim was reported for 2,327 (8%) of the fixed-facility events with the number of victims per event ranging from 1 to 259 (mean = 4 and median = 1). Similarly, at least one victim was reported for 759 (8%) of the transportation events with the number of victims per event ranging from 1 to 65 (mean = 2 and median = 1).The analysis of risk factors for evacuations focused on 39,622 events (30,190 (76%) fixed-facility events and 9,432 (24%) transportation-related events) because information about whether an evacuation was ordered was missing for 144 events. Evacuations were ordered for 2,961 (10%) fixed-facility events with the number of persons evacuated per event ranging from 0 (10 events) to 11,000 (mean = 112 and median = 20). For transportation events, 453 (5%) events involved an ordered evacuation; the number of persons evacuated per event ranged from 0 (3 events) to 8,700 (mean = 163 and median = 20). For fixed-facility events, the area of evacuation was primarily the building(s) or affected part of the building(s) (2,222 events, 75%), while for transportation events, the evacuation zone was primarily a circular area (199, 44%).Emergency chemical releases at fixed facilities involving victims were more likely to occur on weekdays than weekends  . Releases of chlorine (POR = 4.90, 95% CI = 4.07–5.89) were most likely to result in fixed-facility events with victims compared with releases from all other chemical categories while releases of multiple substances from different categories (POR = 7.46, 95% CI = 5.92–9.41) were most likely to result in transportation events with victims.Type of industry was examined as a potential risk factor for victims in fixed-facility events only. Industries classified as personal services were most likely to result in events with victims than were releases not involving these industries (POR = 8.24, 95% CI = 7.20–9.44)  Table . FurtherFixed-facility events with evacuations were most likely to occur in areas described as commercial compared with the referent group "other." Area type was not associated with ordered evacuations for transportation-related events. The release type fire and explosion was most likely to result in fixed-facility events with evacuations compared with spills (POR = 11.90, 95% CI = 7.92–17.89). Releases of chlorine were most likely to result in events with evacuations compared with releases from all other chemical categories for both fixed-facility and transportation-related events (POR = 4.79, 95% CI = 4.03–5.71 and POR = 7.52, 95% CI = 2.93–19.31, respectively).We examined type of industry as a potential risk factor for evacuations in fixed-facility events only. Industries classified as professional services were most likely to result in events with evacuations compared with releases not involving these industries (POR = 7.82, 95% CI = 6.85–8.94) , day of week (weekday), and season (March through May) (p = 0.06); no interaction among temporal variables was indicated for evacuations.The likelihood of an event with at least one victim or evacuation in fixed facilities was examined for substances within industry category Table . The strAlmost 40,000 chemical release events were reported to HSEES during the 6-year period included in this analysis. Approximately 8% of the events resulted in almost 12,000 victims, and approximately 9% involved evacuation of more than 325,000 people. Because the public health consequences of chemical releases can be serious and affect large numbers of people, identifying the risk factors more likely to be associated with hazardous substance releases that result in victims or evacuations is important to target appropriate prevention activities.For fixed-facility events, fire and explosion was the strongest single risk factor for events involving either victims or evacuations. Illegal dumping or deliberate damage also was strongly associated with fixed-facility events resulting in victims. For transportation-related events, explosion was the strongest single risk factor for events involving victims, and fire and explosion was the strongest for events involving evacuations. The observed associations between time of day (0600–2359) and weekday occurrence are likely to be influenced by production intensity, however information on production intensity is not available in HSEES to examine this relationship further. When temporal variables were modeled together, fixed-facility events with victims were more likely to occur on weekday mornings in the spring, which may coincide with the planting season. These results are consistent with a previous analysis that showed that acute hazardous substance releases with victims were more likely during the planting season in Midwestern states .In the stratified analysis of substances within industry category, the strongest association was for acid releases in the agriculture, forestry, and fisheries industry for fixed-facility events involving victims. Previous analyses have shown that acute chemical releases are common in the agricultural industry, nitric acid and sulfuric acid are frequently released in agriculture events, and releases of acids result in significantly higher proportions of releases involving victims ,8,9.Chlorine releases in fixed facilities resulted in victims and evacuations in more industry categories than any other substance. This is consistent with an analysis that found chlorine releases had the greatest significant risk of having events with victims and were almost five times more likely than nonchlorine events to involve evacuations . This isThe HSEES system collected data in only 17 states during 1996–2001. Each state has different reporting requirements for the amount of hazardous substances released that has to be removed, cleaned up, or neutralized; and reporting of events to participating state health departments is not mandatory. Therefore, the total number of events, events with victims, and events with evacuations may be underestimated. However, HSEES is the only federal hazardous substances release database designed specifically to assess and record the public health consequences of hazardous substances emergency events.The HSEES system captures more events than other federal reporting systems, such as the United States Environmental Protection Agency's (EPA) Risk Management Program (RMP) . EPA's RThese results indicate that prevention activities should focus on ways to reduce fire and/or explosion hazards in facilities that store, use, or manufacture hazardous substances such as distributing material safety data sheets to employees, keeping work areas dust free, and storing chemicals away from ignition sources. Previous analyses of HSEES data have shown that human error and equipment failure are the most frequent causes of acute chemical releases ,16-18; rSeveral state health departments participating in HSEES already have developed strategies aimed at reducing releases and injuries associated with chlorine. These activities include distribution of fact sheets to county emergency management agencies, fire departments, other first responders, and industries; and presentations to municipal water directors, engineers, and hotel/motel swimming pool owners and operators.The predictors of public health consequences associated with acute chemical releases presented in this paper are broad categories of characteristics available in the national data. Future analyses of HSEES data may explore industry-specific root causes of events and risk of events with victims or evacuations for specific industries.The results of this analysis should help guide prevention activities aimed at reducing emergency chemical releases and their associated victims and evacuations. Attention should focus on preventing fires and explosions, releases from illegal dumping and deliberate damage, and acid releases in the agricultural industry. Efforts should continue to educate industry, first responders and other users about the potential hazards of chlorine.ATSDR, Agency for Toxic Substances and Disease RegistryCI, confidence intervalEPA, Environmental Protection AgencyHSEES, Hazardous Substances Emergency Events SurveillancePOR, prevalence odds ratioRMP, Risk Management ProgramThe author(s) declare that they have no competing interests.PZR led the writing of the manuscript and assisted with the statistical analysis. WAW led the statistical analysis and assisted with the writing of the manuscript. WEK conceived of the manuscript and participated in its writing and interpretation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Poring over the vast amount of sequence and genetic information now available for many organisms, scientists frequently encounter what appear to be redundant biochemical pathways. Redundant pathways take the same starting material and transform it into the same product, but through different routes. Why should cells maintain redundant pathways?Methylobacterium extorquens, a bacterium that can grow on organic molecules with a single carbon atom such as the alcohol methanol. Bacteria in this species first oxidize methanol into formaldehyde, then use formaldehyde to make serine—the entry point for the synthesis of many of the cell's building blocks—via two apparently redundant pathways. The short pathway is a direct (non-enzymatic) reaction of formaldehyde with tetrahydrofolate to make methylene-tetrahydrofolate, which donates a single carbon atom for serine synthesis. A hypothesized long pathway could also lead to methylene-tetrahydrofolate through a long series of enzymatic reactions, one of which consumes energy.An interesting case is that of Christopher Marx and colleagues now demonstrate that the bacteria modulate their use of each pathway during the course of acclimation to growth on methanol. In the process the authors offer new insights into the bacteria's rapid disposal of formaldehyde, a toxic chemical that would pickle cells in a minute if it was allowed to accumulate.The authors noticed that the short pathway transferred both hydrogen atoms of formaldehyde to the serine molecule while the long pathway transferred only one. If they fed bacteria methanol in which hydrogen had been replaced with its heavier form, deuterium: the resulting serine was slightly heavier than normal, as a result of acquiring one—or two—deuterium atoms.By measuring the ratio of the two serine forms, Marx and colleagues inferred the relative contribution of the two pathways to serine synthesis. The long pathway dominated, accounting for eight times more serine than the short pathway, when cells first encountered methanol. But the situation was reversed after the cells acclimated to methanol: then the short pathway produced 15 times more serine than the long pathway.14C). Although the relative contribution of the long pathway decreased during ramping-up to methanol growth, the absolute amount of formaldehyde that flowed through it increased 8-fold within the first half of the transition, and then decreased.The authors also measured absolute amounts of formaldehyde processed by each pathway, using methanol marked with a heavy form of carbon  route to serine production (and hence growth), but one that is slower to reach its cruising speed. Thus, the cells use these two formaldehyde assimilation routes like a driver uses the transmission of a car: start with powerful low gears when first accelerating, and shift to more efficient gears once hurtling down the road.The combination of both pathways represents an elegant solution to the problem of growth in toxic environments and provides a useful paradigm for detoxification in medical and environmental contexts.


Autism is a neurobehavioral spectrum of phenotypes characterized by deficits in the development of language and social relationships and patterns of repetitive, rigid and compulsive behaviors. Twin and family studies point to a significant genetic etiology, and several groups have performed genomic linkage screens to identify susceptibility loci.We performed a genome-wide linkage screen in 158 combined Tufts, Vanderbilt and AGRE (Autism Genetics Research Exchange) multiplex autism families using parametric and nonparametric methods with a categorical autism diagnosis to identify loci of main effect. Hypothesizing interdependence of genetic risk factors prompted us to perform exploratory studies applying the Ordered-Subset Analysis (OSA) approach using LOD scores as the trait covariate for ranking families. We employed OSA to test for interlocus correlations between loci with LOD scores ≥1.5, and empirically determined significance of linkage in optimal OSA subsets using permutation testing. Exploring phenotypic correlates as the basis for linkage increases involved comparison of mean scores for quantitative trait-based subsets of autism between optimal subsets and the remaining families.positive correlations of linkage between the 19p locus and 17q, between 19p and 6q, and between 7q and 5p. While potential phenotypic correlates for these findings were not identified for the chromosome 7/5 combination, differences indicating more rapid achievement of "developmental milestones" was apparent in the chromosome 19 OSA-defined subsets for 17q and 6q. OSA was used to test the hypothesis that 19p linkage involved more rapid achievement of these milestones and it revealed significantly increased LOD* scores at 19p13.A genome-wide screen for autism loci identified the best evidence for linkage to 17q11.2 and 19p13, with maximum multipoint heterogeneity LOD scores of 2.9 and 2.6, respectively. Suggestive linkage (LOD scores ≥1.5) at other loci included 3p, 6q, 7q, 12p, and 16p. OSA revealed Our results further support 19p13 as harboring an autism susceptibility locus, confirm other linkage findings at 17q11.2, and demonstrate the need to analyze more discreet trait-based subsets of complex phenotypes to improve ability to detect genetic effects. Autism OMIM # 209850) is a neurobehavioral disorder involving deficits in language and social abilities and patterns of repetitive behaviors, restricted interests and resistance to change. The most recent estimate of population prevalence for the broader autism spectrum indicates a rate of 34/10,000 (~1/300) 09850 is -8, and tGenome-wide screens of multiplex autism families for susceptibility loci -22 have We report a second generation 10-cM microsatellite-based genomic screen of multiplex autism families. The dataset for this screen includes 71 families recruited by the Tufts/New England Medical Center, a well-characterized set of 85 families from the Autism Genetics Resource Exchange (AGRE), and 2 families from Vanderbilt University. Several sites of suggestive linkage are identified, although none meet criteria for genome-wide significance. The loci with greatest support for linkage were 17q11.2 and 19p13; the latter site demonstrated significantly increased allele-sharing when the Ordered-Subset Analysis (OSA) algorithm was employed using a quantitative trait-based autism phenotypic subset related to specific "developmental milestones" as a covariate to rank families.The demographics for the 158 family dataset comprising the studies in this report are shown in Table DNA from Tufts and Vanderbilt samples was obtained from peripheral blood or immortalized lymphoblastoid cell lines using the PureGene Kit (Gentra Systems). While a minority of families from the Tufts/NEMC cohort had been genotyped previously , both neGenotype data for each chromosome underwent thorough error detection and genotype confirmation. Initially, data were tested for Mendelian inconsistencies using PEDCHECK  and RELPAllele frequencies were estimated using genotype data from all unrelated individuals in the combined dataset, consisting of more than 300 chromosomes. Allele frequencies were compared with available data from other Caucasian populations, and no significant differences were observed (data not shown). The LAPIS program of the PEDIGENE system  was usedSpairs scoring function as recommended by McPeek [Linkage was analyzed using both model-dependent and model-independent methods. For autosomes, two-point and multipoint heterogeneity LOD (HLOD) scores were calculated under both dominant and recessive models using Allegro . Diseasey McPeek . NPL scoy McPeek  and FASTy McPeek  to calcuth percentile of the resulting distribution.The nonparametric genome-wide significance threshold ,49 for lOSA  identifi(1) To walk unaided; (2) to sit unaided on flat surface; (3) age of first single words; (4) age of first phrase; (5–6) acquisition of bladder control: daytime, night; (7) acquisition of bowel control."To explore potential genetic interaction or other genetic correlations between sites of main effect (i.e. suggestive linkage), OSA was applied using family-specific LOD scores as the covariate trait. Families were ranked according to the family-specific LOD score at peak sites demonstrating LOD scores ≥1.5. Allele-sharing analysis was performed for the other six chromosomes using the OSA algorithm. For instances of empirically significant increases in evidence for linkage, we explored the nature of the genetic correlation to ask whether it reflected clinical correlations in the respective subsets. We employed ADI-based factor subsets, identified by principal components analyses of ADI/ADI-R items, to represent putative phenotypic subsets in autism ,56. The Analysis of the "developmental milestones" factor as a potential phenotypic subset related to the autism linkage correlations was performed by applying the OSA algorithm. We used "developmental milestones" family means, normalized via SAS and Box-Cox transformation procedures, as an ascending ranking covariate. LOD* scores were calculated according to the OSA algorithm, and the resulting increase in linkage achieved with the OSA-determined family subset was analyzed through permutation testing.Approval for these studies was granted by the respective Institutional Review Boards at Tufts University School of Medicine/New England Medical Center and Vanderbilt University Medical Center. Additionally, all studies were performed with informed consent provided by the families participating in the research.Seven chromosomes revealed one or more regions of linkage with a model-dependent or model-independent LOD score ≥1.5 Figure . Data prSLC6A4) locus, long considered to be an attractive functional candidate gene for autism and other neuropsychiatric conditions. Figure For 17q11.2, peak linkage was observed at 53 cM, corresponding to marker D17S1294  and 6 (30 families) for the "developmental milestones" cluster. The families in the optimal OSA subset have lower scores and therefore are more rapidly achieving developmental milestones. A similar procedure for the chromosome 7-based subset revealed no obvious differences in any of the factors (data not shown).To directly test the hypothesis that chromosome 19 linkage was related to reduced affection for the "developmental milestones" factor, we performed an OSA analysis in which families were ranked in ascending order based on mean values for the milestones factor score. Figure We have presented evidence in support of autism susceptibility loci on chromosomes 17q and 19p. Our results suggest that the 19p locus is related to a phenotypic profile involving a more rapid achievement of particular "developmental milestones". Features indexed in this ADI-based factor are: (1) ability to walk unaided; (2) ability to sit unaided on a flat surface; (3) age of first single words; (4) age of first phrase; (5–6) acquisition of bladder control: daytime and night; and (7) acquisition of bowel control. Analyses leading to this conclusion also showed positive genetic correlations between optimal OSA-defined subsets contributing to linkage at 19p13 and increases in linkage at loci on 17q21 and 6q23. A similar positive genetic correlation was shown for chromosomes 7q and 5p, however this observation lacks evidence of an underlying phenotypic relationship based on available ADI variable clusters. While the increase in linkage at 17q21 was not empirically significant, the differences in "milestone" score means between the optimal chromosome 19 subsets seen for both chromosomes 17 (52 families) and 6q (30 families) were significant. These exploratory data led to the significant finding of increased linkage in the single direct test of our hypothesis concerning the phenotypic correlation related to chromosome 19 linkage.Despite the significance of the final results on 19, we remain cautious in the interpretation of the overall results. As with a number of other genomic screens in autism, no single main effect locus achieved genome-wide significance. Support for a number of these loci, particularly at 17q11.2 and 19p13 comes from similar suggestive linkage in other genomic screens for autism. Although not all screens detect these loci (not an uncommon finding in linkage studies for complex genetic disorders), the evidence is strong regarding an effect at 19p, within 10 cM of our peak: (1) Shao et al reported an MMLS = 1.21 and an MLOD = 1.38 ; (2) theSLC6A4 [SLC6A4 gene on chromosome 17q11.2. Our own more preliminary analysis of linkage in this region with a highly overlapping dataset to that in the current study, revealed very similar results [nd-stage screen [Similarly, several groups have reported evidence for linkage at 17q11. The recently published AGRE follow-up genomic screen identified an MLS of 2.83 near SLC6A4 . A genomSLC6A4 . An IMGSSLC6A4  reported results . Given oe screen , howeverITGB3) locus, which was identified recently from a genome-wide quantitative trait locus (QTL) association screen for platelet serotonin levels [The 17q21 locus is worth further consideration. Our data support the premise that the adjacent linkage peaks represent distinct loci and are not an artifact of primary linkage at 17q11.2. The evidence for linkage at 17q21, while weaker than that at 17q11.2 only 16 cM centromeric, specifically showed an, albeit nonsignificant, interlocus correlation with 19p13 linkage. Linkage at 17q11.2 in this subset of families actually decreases slightly. Of particular interest is the fact that the distal region harbors the integrin β3 (n levels . We see The other "suggestive" (LOD ≥ 1.5) loci reported here have also been detected in other genome-wide scans for autism loci. A broad region of 7q has been detected in most screens ,25,27,28that linkage to 19p13 was related to a more rapid achievement for specific milestones. We tested this hypothesis with a single analysis revealing an empirically significant increase in linkage at 19p13. Our results of autism linkage and its increase using an ascending milestone score covariate in OSA, taken in the context of replicated observations of suggestive linkage by other groups, strengthens support for the presence of an autism gene at this site. In the end, ultimate interpretation will rely upon replication of these phenomena with independent samples to confirm these observations.The application of OSA to detect putative interlocus correlations between the 19p13 and 17q21, 19p13 and 6q23, and between 7q35 and 5p are limited to some degree in significance by their highly exploratory and hypothesis-generating nature. Given the number of comparisons between loci, and the number of comparisons between optimal subset pairs (on 19p or 7q) for the traits means, the potential for type I error is increased. Therefore our interpretation must be cast alongside appropriate caveats. Nevertheless, the multiple exploratory comparisons generated a hypothesis: Finally, our results highlight the utility of using trait-based subsets of autism to identify putative susceptibility loci for this complex disorder. We and others have hypothesized a likely increased specificity of individual risk genes and corresponding alleles for traits or subphenotypes comprising the broader autism spectrum. Therefore methods such as OSA with power to identify more homogeneous samples and QTL (quantitative trait locus) linkage and association analyses should provide greater sensitivity in the discovery of disease genes in the context of locus and clinical heterogeneity. Additionally, OSA or other forms of conditional linkage analyses, have the ability to uncover potential interactions between loci, an important concept since the inherent interdependence of proteins in common pathways or networks acting during development and normal neuronal function could be easily imagined to act genetically in concert with one another.We report evidence to support linkage of autism to 17p11.2 and 19p13. Exploratory analyses to test for correlations between suggestively-linked loci, using the OSA method revealed positive correlations of linkage (i.e. in overlapping families) between 7q and 5p, 19p and 6q, and possibly 19p and 17q22, distal to peak linkage at 17q11.2. Comparing mean scores for ADI-derived factor traits from families above and below the OSA-defined split maximizing linkage, suggested a positive correlation between 19p13 linkage and a more rapid achievement of "developmental milestones" as measured by items in this cluster of ADI variables. We tested this hypothesis by applying OSA with descending "developmental milestone" scores as the ranking covariate, and detected an empirically-significant increase in linkage to 19p13. These findings further support evidence for an autism susceptibility locus in 19p13 and underscore the utility in applying trait subsets in complex disorders to identify genetic risk factors.The authors declare that they have no competing interests.JLM and LMO were responsible for conducting the genome-wide linkage analyses; JLM conducted all OSA analyses, was largely responsible for coordinating and executing the bulk of the reported studies, was key to drafting and editing the manuscript text, and in preparing the figures. CL and JLH provided input into the design and interpretation of statistical results, and provided guidance for conducting the genome-wide simulations required to determine the significance threshold. JLH established the Core-based infrastructure in the Center for Human Genetics Research that facilitated this work and provided very helpful input into the development of the manuscript. SEF developed, with her group, the ADI-based clusters so crucial to examining phenotypic subsets in this paper, which she helped to edit. SEF was responsible for overseeing recruitment and the phenotypic assessment of families from her research group, then at Tufts/NEMC. GC is the clinical coordinator at the Vanderbilt site and oversees ascertainment, recruitment and detailed phenotypic assessment of affected individuals. KG is the Vanderbilt data coordinator and is responsible for management and oversight of family information, pedigree data, and status of DNA, blood or cell line samples for family members. She is directly responsible for preparation of Table The pre-publication history for this paper can be accessed here:


Anyone who uses a word processor is likely thankful for the spell checker program. But that autocorrect function can introduce errors, “correcting” the spelling of words to fit its stored repertoire, which is decidedly limited. Take that one step further and imagine a rogue program that destroys the coherence and meaning of your prose by swapping out one letter for another throughout the document. That's the situation retroviruses like the human immunodeficiency virus (HIV) face during the course of their infectious cycle, when a protein encoded by the host genome slips into the virus, mutates the virus's genetic material, and alters the viral genome.APOBEC3G, belongs to a family of primate genes that produce enzymes (in this case, APOBEC3G) that “edit” DNA and RNA, by slipping into viral particles and inducing mutations that replace one base (cytosine) with another (uracil) as the virus undergoes reverse transcription in the host cell's cytoplasm. The edited virus fails to replicate. HIV, in turn, generates a protein called Vif that binds to the APOBEC3G enzyme and targets it for degradation, thereby eliminating its antiviral activity.The gene, Since the protein-binding regions that govern these interactions have a direct effect on the fitness of both virus and host, one would expect to see the proteins angling for advantage, with Vif maximizing its ability to recognize APOBEC3G and APOBEC3G doing its best to evade Vif. Such battles are thought to result in frequent mutations that alter the amino acids involved in the interaction; the perpetuation of such advantageous mutations is called positive selection.PLoS Biology, Sara Sawyer, Michael Emerman, and Harmit Malik investigate the genetic roots of this battle for evolutionary advantage and find something surprising. As predicted, the APOBEC3G gene is under strong positive selection. But that selection appears to predate the existence of HIV-type viruses.In this issue of APOBEC3G evolution, Sawyer et al. analyzed the gene from twelve primates—New World monkeys, Old World monkeys, and great apes, including humans—spanning 33 million years of evolution. Most of the primate lineages showed evidence of positive selection, indicating that the gene has been under pressure to adapt throughout the history of primate evolution. But viruses like HIV have been found in only five of the primates studied—three African monkeys, chimpanzees, and humans—and appear to be at most one million years old. And HIV infection in human populations is too recent to account for the positive selection of APOBEC3G in humans—so what has been fueling APOBEC3G's rapid evolution?To characterize the selective pressures on APOBEC3G has been constant over the course of primate evolution suggests that another force is also acting on the gene. Sawyer et al. propose that this force is most likely occurring in germline cells (sperm and egg precursors), which also produce high levels of APOBEC3G and can pass mobile genetic elements on to the next generation. Despite being non-infectious, these elements increase their own copy number in the host genome, moving from one part of the genome to another. The human genome is littered with such “retrotransposons,” and it is these mobile genetic elements, the authors conclude, that likely antagonize APOBEC3G.APOBEC3G and Vif interact in T-cells, but the fact that selective pressure on One class of retrotransposons, called human endogenous retroviruses, acts in many ways like foreign retroviruses. A retrovirus emanating from one's own genome poses less of an immediate threat than a retrovirus like HIV. But the constant efforts of the endogenous retrovirus to “jockey for evolutionary dominance,” the authors conclude, could eventually take a toll and would be expected to provoke efforts to contain it. And it may be that this ancient intragenomic conflict endowed APOBEC3G with the means to do battle with foreign retroviruses like HIV.APOBEC human genes appear to be engaged in similar conflicts. Combined with the finding that rodents have only one APOBEC3G gene and that five out of the six human APOBEC3 genes have been under positive selection, these results suggest that this gene family expanded in mammalian evolution as a means of defending the germline from the promiscuous intrusions of mobile genetic elements.Sawyer et al. also found evidence that five other