Aβ peptide interactions with isoflurane, propofol, thiopental and combined thiopental with halothane: A NMR study

Aβ peptide is the major component of senile plaques (SP) which accumulates in AD (Alzheimer's disease) brain. Reports from different laboratories indicate that anesthetics interact with Aβ peptide and induce Aβ oligomerization. The molecular mechanism of Aβ peptide interactions with these anesthetics was not determined. We report molecular details for the interactions of uniformly ¹⁵N labeled Aβ40 with different anesthetics using 2D nuclear magnetic resonance (NMR) experiments. At high concentrations both isoflurane and propofol perturb critical amino acid residues (G29, A30 and I31) of Aβ peptide located in the hinge region leading to Aβ oligomerization. In contrast, these three specific residues do not interact with thiopental and subsequently no Aβ oligomerization was observed. However, studies with combined anesthetics (thiopental and halothane), showed perturbation of these residues (G29, A30 and I31) and subsequently Aβ oligomerization was found. Perturbation of these specific Aβ residues (G29, A30 and I31) by different anesthetics could play an important role to induce Aβ oligomerization.     

Taking U out, with two nucleases?

Background  

REX1 and REX2 are protein components of the RNA editing complex (the editosome) and function as exouridylylases. The exact roles of REX1 and REX2 in the editosome are unclear and the consequences of the presence of two related proteins are not fully understood. Here, a variety of computational studies were performed to enhance understanding of the structure and function of REX proteins in Trypanosoma and Leishmania species.     

Some Salt with Your Statin,Professor?

We know that clinical trials sponsored by the pharmaceutical industry are likely to exaggerate benefit and minimise harms. But do these biases extend to their sponsorship of non-human animal research? Using systematic review and meta-analysis Bero and colleagues show that, in the case of statins, things are a little more complicated. While the conclusions of industry-sponsored studies were indeed more enthusiastic than warranted by their data, the data themselves painted a picture more conservative than was seen in non-industry-sponsored studies. This behaviour is consistent with maximising the return on investment, seeking robust data before embarking on a clinical trial, and, once that investment has been made, making every effort to “prove” that the drug is safe and effective if this is at all credible. The findings suggest that there is something different about industry-sponsored non-human animal research, perhaps reflecting higher standards than is the case elsewhere. Perhaps the academic community can learn something from our colleagues in the commercial sector.It is now pretty clear that, in clinical trials, sponsorship from the pharmaceutical industry is associated with substantial and important overstatement of how effective drugs are, and with understatement of adverse effects [1]. Of course, these are average effects, and so are insufficient to label the whole industry bad. Nonetheless, there are many examples where industry has been shown to seek to subvert rational interpretation of trial data to influence guideline development and prescribing behaviour [2]–[4]. These examples lead to the reasonable conclusion that findings from trials sponsored by the pharmaceutical industry need to taken with more salt than is probably good for you.What then of other research used to inform the drug development process? What of the in vitro and in vivo non-human research supported by industry, either in companies'' own laboratories or that companies fund in contract research organisations or in academic collaborations? Are the findings of such studies credible? And how do those findings compare with “proper” research conducted by dispassionate academics?These are important questions, but how could we find this stuff out? In the same way that it would be difficult to conduct a randomised controlled trial of the effect of living in Scotland on your chance of having a stroke, it is difficult to do an experiment to test whether the funding source for a study influences the outcome. We have to rely on observational (rather than experimental) research, and we need to be much more cautious in our approach and in our conclusions.Over the last few years there has been a big increase in the use of such an observational approach to better understand the strengths and weaknesses of different research domains. The Cochrane Collaboration began as an attempt to give reliable summaries of the effectiveness of treatments in human clinical trials [5], but along the way the data collected have also allowed investigation of whether studies with certain characteristics tended to give overstatement or understatement of these summary treatment effects [6]. The insights arising from this approach, and the improvements in trial design that they have driven, are just as important as the improved information to guide treatment decisions. This approach has been used by others—notably Lisa Bero, the senior author of the research article presented here—in a series of important papers that identified the prevalence and impact of funding bias in human research [7],[8].Those wishing to study, and to improve, other research domains such as non-human animal research have been able shamelessly to borrow from the experience of the Cochrane Collaboration. Using a systematic approach to data retrieval we can assemble an unbiased cohort of relevant studies, then observe associations between different aspects of experimental design and the magnitude of the effects reported. What we''re looking for are design features that are consistently associated with either under- or overestimation of biological effects.Of course, meta-analyses of clinical trial data put together a small number of large studies measuring a common treatment effect, whereas in animal studies there is usually a large number of small studies measuring different effects (dose, stage of illness, different animals), which means the approach used has to be adjusted slightly, but still, the approach has been fruitful.For a large number of non-human animal disease models, studies at risk of bias (for example, those without randomisation or blinding) give larger estimates of treatment effects [9]–[13]; the majority of studies are at risk of bias [9]–[14]; and journal impact factor is no guarantee of low risk of bias [15]. These findings influenced the development of reporting standards for stroke [16] and non-human animal research more generally [17],[18], and these are beginning to make an impact.One difficulty in using meta-analysis is in working out how to combine different outcome measures, often from different animals. A 0.1-mm increase in aortic arch atheroma is probably less important in a Scot than it is in a mouse, so we need to transform data onto a common scale. In standardised mean difference (SMD) meta-analysis, the effect is standardised to the observed variance [19]. Because—in large studies at least—this variance is a property of the biology being studied rather than of the scale being used, it allows effects to be converted to a common scale. So, by way of an example: in 2012 the variance of the monthly average temperature across 258 weather stations in California was 12.55°F, or 6.98°C—from which we can calculate that 1°C is the same as 1.80°F, or 0.14 standardised units, and so we have a common scale.While this approach is very useful in clinical meta-analyses (where the large number of participants in each group allows a precise estimate of the population variance), it becomes less useful where group size is small, because here the observed variance is a less precise estimate of the population variance. This introduces a measurement error to the conversion between different scales.Further, this observed variance represents a combination of underlying biological variation in the phenomena being measured and of variation arising from measurement error and from the way the experiment was performed. Experiments with low measurement error and good protocol compliance will therefore have lower aggregate variance than those with high measurement error and poor protocol compliance. Since the variance is the denominator in the calculation of the size of differences between groups, any given effect size will be artificially larger in studies with low measurement error and experimental variability.The demonstration that experiments with low methodological quality can give inflated estimates of treatments effects, and that most experiments appear to be of low methodological quality, leads to the question of who might be the worst offenders. Since clinical trials sponsored by the pharmaceutical industry seem to be at greater risk of bias than others, a lazy assumption might be that their non-human animal research is similarly confounded, as they seek to rush compounds to market to maximise profitability.However, a few straws in the wind hint this might not be the case. One way companies identify drug targets is by reading what''s out there in the literature and, if something looks interesting, seeking to replicate the findings. Bayer scientists found inconsistencies in 43 of 65 studies when they tried to replicate them in-house [20]. Scientists in the haematology and oncology departments at Amgen were able to replicate findings in only six out of 53 publications identified as “landmark” studies [21]. When the ALS Therapy Development Institute tried to replicate published findings of drug efficacy in the superoxide dismutase mouse model of motor neuron disease (amyotrophic lateral sclerosis), not one of seven interventions retained efficacy [22]. Implementation of good laboratory practice standards is much more advanced in industry labs, and for some types of experiments these standards are a legal requirement. Indeed, a scientific researcher was recently jailed in Scotland for research fraud [23]. So, could it be that industry-sponsored research is actually more rigorous than academic research?Taking the example of statin treatments for atheroma, David Krauth, Andrew Anglemyer, Rose Philipps, and Lisa Bero address this issue head-on [24]. Using systematic review they identified non-human animal studies describing the efficacy of statins. Their methodology is secure, with an a priori analysis plan, clear inclusion and exclusion criteria, and duplicate extraction of key variables from identified publications. They found low levels of reporting of measures known to reduce the risk of bias, with blinded assessment of outcome reported in only 22 of 49 studies, and no studies reporting full randomisation or a sample size calculation. Reassuringly, the quality of reporting seems to have improved somewhat since publication of the ARRIVE guidelines in 2010. However, there is still clearly a long way to go.On the question of the influence of the study sponsor, Bero and colleagues identified 19 studies sponsored in whole or part by industry, 28 sponsored by non-industry sources, and 16 with no statement of sponsorship or a statement of no sponsorship. Focussing on those studies where sponsorship status was known, they found that the results of nine of 19 industry-sponsored studies (43%) and 18 of 28 non-industry-sponsored studies (72%) supported the efficacy of statins. This finding was confirmed in a subset of 38 studies with sufficient data to allow meta-analysis; statins were reported to improve outcome by 0.73 SMD units in industry-sponsored studies, while in studies with other sponsorship the improvement was 1.99 SMD units. This difference is highly significant—I calculate an excess of efficacy in non-industry-sponsored studies of 173% (95% confidence interval 52% to 293%). Put simply, studies with non-industry sponsorship report that statins are almost three times more effective than do industry-sponsored studies.As interesting, however, is the analysis of the interpretation placed on the findings in each of the included studies. Of 19 industry-sponsored studies, the conclusion of 18 favoured the use of statins (95%), while of 28 non-industry-sponsored studies, only 21 did so (75%). This is striking for two reasons: first, in both cohorts the conclusion appears to be more enthusiastic than the findings presented, and second, this phenomenon appears to be much more marked in studies with industry sponsorship.So what''s going on? Of course, these observed differences may be due to some other, unmeasured difference between the contributing studies, but the analyses were prespecified and such a confound appears unlikely. If industry-sponsored studies were of consistently larger variance, then the effect sizes observed would appear smaller in SMD units, but there is no reason to suspect that this was the case here.It does therefore appear that findings from research sponsored by industry are more conservative than those sponsored by non-industry sources, but the interpretation of those data is, in contrast, less conservative. Why might this be?In a series of univariate analyses the authors examined the impact of three factors—randomisation, blinding, and accounting for all animals—that might increase the risk of bias. Even when these were taken into account, non-industry-sponsored studies gave significantly higher estimates of efficacy, implying that some other factors were responsible. This might happen if “randomisation” and “blinding” meant different things in industry-sponsored studies, or through the impact of some other, unmeasured risk of bias, or through some gestalt of industry-sponsored studies that is not described by the variables tested. Alternatively, academic studies exploring pathophysiology might chose circumstances that maximise the observed effect size, to give greater statistical power to experiments testing inhibition of those effects.In my view it is likely that the impact of approaches to research management and the regulatory environment that apply to some parts of industry—particularly standards for internal reporting—extends to most of the non-human animal research activity with which they are involved, whether or not it is performed in-house. That is, non-human animal work sponsored by industry is likely to be performed and reported to a higher quality, and to be at lower risk of bias, than work sponsored by others. This would explain the difficulty industry has in replicating the results of research conducted in academic labs. However, the interpretation, or “spin”, with which industry-sponsored work is presented does appear to be an issue, with exaggeration of the conclusions to favour the drug being tested.This makes sense—for industry there is a clear financial interest in being absolutely secure in the non-human animal data for a compound before embarking on a clinical trial, so there is a real motivation to get the preclinical data as good as they can be. Clinical trials are expensive, and so it is worth investing much time and effort, and perhaps even funding multicentre “phase 3” animal studies [25]–[27], to maximise the prospects for success. But when that money has been spent (and for statins it largely has been), the motivation is to present an analysis of the available data that is most supportive for clinical use. So, if a drug is a turkey, try to find that out before spending a fortune taking it to clinical trial—and if it''s too late for that, try to convince everyone that the non-human animal and clinical trial data supporting an efficacy for Meleagris gallopavo (commonly known as the wild turkey) are more convincing than they might at first appear.In contrast, academic researchers are rewarded not for the marathon but for the sprint—for a high-impact publication describing a part of the jigsaw, not for the body of work that shows the whole picture. To them, substantial efficacy in a single study is, in some respects, an end rather than a beginning.Bero and colleagues have made an important contribution; their findings suggest that academic researchers might learn good practice in the management, conduct, and reporting of non-human animal research from colleagues in industry, and reinforces the importance for readers of research reports to focus on methods and data rather than on abstracts and conclusions.     

What's wrong with novel ecosystems,really?

The novel ecosystems concept has gained much traction in the restoration community. It has also drawn the ire of several prominent ecologists and is the focus of an ongoing debate. We consider three key aspects of this debate: irreversible thresholds, non‐native species, and the hybrid state. Irreversible thresholds have been acknowledged in restoration for years, but predicting when a threshold will be crossed and the degree of reversibility is problematic. Oftentimes reversibility is a function of multiple factors, such as cost and public support. In this sense, a novel ecosystem is not an alternate state but a decision. The need for pragmatism regarding control of non‐natives has also long been recognized in restoration circles. Proponents of the novel ecosystem idea adopt this pragmatism by recommending that management decisions be based on impacts conferred by species in altered ecosystems, regardless of their origin. The concept of a hybrid state has proven difficult to operationalize. We suggest that rather than trying to identify the boundary between hybrid and novel states, ecosystems exist on a gradient of alteration. We offer a decision tree for restoration action that integrates aspects of novel ecosystems with other perspectives in modern restoration ecology. We conclude that the idea of novel ecosystems, though not perfect, deserves a place under the “big tent” of restoration that includes efforts to return fully to a reference state, as well as strategies for reinstating lost ecological processes and enhancing ecosystem services in transformed landscapes where such a return is deemed infeasible.     

Meteorological factors are associated with hemorrhagic fever with renal syndrome in Jiaonan County,China, 2006–2011

This study examined the effect of meteorological factors on the occurrence of hemorrhagic fever with renal syndrome (HFRS) using a generalized additive model with penalized smoothing splines in Jiaonan, China, from 2006 to 2011. The dose–response relationship was first examined, and then the association between daily meteorological variables and HFRS occurrence was investigated according to the dose–response curves. There were two linear segments in the temperature–HFRS relationship curve. When daily temperature was lower than 17 °C, a positive association was found [with excessive risk (ER) for 1 °C increase on the current day being 2.56 %, 95 % confidence interval (CI): 0.36 % to 4.80 %]. An inverse association was found when daily temperature was higher than 17 °C [ER for 1 °C increase on the current day was ?12.82 % (95 % CI: ?17.51 % to ?7.85 %)]. Inverse associations were observed for relative humidity [ER for 1 % increase on lag day 4 was ?1.21 % (95 % CI: ?1.63 % to ?0.79 %)] and rainfall [ER for 1 mm increase on lag day 1 was ?2.20 % (95 % CI: ?3.56 % to ?0.82 %)]. Meteorological factors might be important predictor of HFRS epidemics in Jiaonan County.     

Treatment with PBI-4050 in patients with Alström syndrome: study protocol for a phase 2, single-Centre,single-arm,open-label trial

Background

Alström syndrome (ALMS) is a very rare autosomal recessive monogenic disorder caused by a mutation in the ALMS1 gene and characterised by childhood onset obesity, dyslipidaemia, advanced non-alcoholic fatty liver disease, diabetes and extreme insulin resistance. There is evidence of multi-organ fibrosis in ALMS and severity of the disease often leads to organ failure with associated morbidities, resulting in reduced life expectancy. There are no specific treatments for this disease, and current management consists of only symptomatic therapies. PBI-4050 is a new molecular entity with demonstrated anti-inflammatory and anti-fibrotic activities in preclinical models, including animal models of human diseases characterized by progressive fibrosis in the kidney, heart, liver and lungs. Moreover, completed Phase 2 studies in type 2 diabetes mellitus with metabolic syndrome and idiopathic pulmonary fibrosis further support the anti-inflammatory and anti-fibrotic activity of PBI-4050. Together, these data suggest that PBI-4050 has the potential to treat the pathological inflammatory and fibrotic features of ALMS. The aim of this study is to evaluate the safety and anti-inflammatory & anti-fibrotic activities of PBI-4050 in subjects with ALMS.

Methods

This is a Phase 2, single-centre, single-arm, open-label trial. A total of 18 patients with ALMS will be enrolled to receive PBI-4050 at a total daily oral dose of 800?mg for an initial 24?weeks with continuation for an additional 36 or 48?weeks. Standard assessments of safety include adverse events, clinical laboratory tests, vital signs, physical examination and electrocardiograms. Efficacy assessments include adipose tissue biopsy, hyperinsulinaemic-euglycaemic glucose clamp, adipose tissue microdialysis, liver transient elastography, liver and cardiac magnetic resonance imaging, and laboratory blood tests.

Discussion

This is the first clinical study of PBI-4050 in subjects with ALMS. Given the rarity and complexity of the disease, a single-centre, single-arm, open-label design has been chosen to maximise subject exposure and increase the likelihood of achieving our study endpoints. The results will provide valuable safety and preliminary evidence of the effects of PBI-4050 in ALMS, a rare heterogeneous disease associated with progressive fibrosis and premature mortality.

Trial registration

The trial is registered on ClinicalTrials.gov (Identifier; NCT02739217, February 2016) and European Union Drug Regulating Authorities Clinical Trials (EudraCT Number 2015–001625-16, Sept 2015).

     

NMR assignment of polymerase β labeled with 2H, 13C, and 15N in complex with substrate DNA

DNA Polymerase β is a multifunctional enzyme involved in base excision repair of nuclear DNA in vertebrate cells. We present here the first assignments of the full-length protein (335 residues, 39 kDa) in the presence of a double gap—double hairpin DNA (22 nucleotides, 7 kDa).     

Golimumab in patients with active rheumatoid arthritis after treatment with tumor necrosis factor α inhibitors: findings with up to five years of treatment in the multicenter,randomized, double-blind,placebo-controlled,phase 3 GO-AFTER study

IntroductionThe aim of this study was to assess long-term golimumab therapy in rheumatoid arthritis (RA) patients who discontinued previous tumor necrosis factor-α (TNF)-inhibitor(s).MethodsPatients enrolled into this multicenter, randomized, double-blind, placebo-controlled study of active RA (≥4 tender, ≥4 swollen joints) received placebo (Group 1) or golimumab 50 mg (Group 2) or 100 mg (Group 3) injections every 4 weeks. Patients in Groups 1 and 2 with inadequate response at week 16 escaped to golimumab 50 and 100 mg, respectively. At week 24, Group 1 patients crossed-over to golimumab 50 mg, Group 2 continued golimumab 50/100 mg per escape status, and Group 3 maintained dosing. During the long-term-extension (LTE), golimumab 50 mg could be increased to 100 mg, and 100 mg could be decreased to 50 mg. Data through 5 years are reported for all patients (safety) and patients using methotrexate (efficacy, intention-to-treat (ITT) analysis with last-observation-carried-forward for missing data and non-responder imputation for unsatisfactory efficacy discontinuations).ResultsIn total, 459 of 461 randomized patients received the study agent, 304 of whom were methotrexate-treated and included in efficacy analyses. Through week 256, the proportions of methotrexate-treated patients achieving American-College-of-Rheumatology (ACR) responses were 37.6% to 47.0% for ACR20, 21.4% to 35.0% for ACR50, and 7.8% to 17.0% for ACR70 response across randomized groups. Golimumab safety through week 268 was generally consistent with that at week 24 and week 160 and other anti-TNF agents.ConclusionsIn some patients with active RA discontinuing previous TNF-antagonist therapy, golimumab safety and efficacy, assessed conservatively with ITT analyses, was confirmed through 5 years.

Trial registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00299546","term_id":"NCT00299546"}}NCT00299546. Registered 03 March 2006.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0516-6) contains supplementary material, which is available to authorized users.     

Out with the old,in with the new? Comparing methods for measuring protein degradation

Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library products by flow cytometry [Yen et al. (2008) Science 322, 918]. While no method is perfect, we conclude that chimeric gene reporter approaches, though powerful, should be applied cautiously, due principally to GFP (or other reporter tag) interference with protein organelle targeting or incorporation into macromolecular assemblies, both of which cause spuriously high degradation rates.     

Plasma visfatin,associated with a genetic polymorphism −1535C > T,is correlated with C-reactive protein in Chinese Han patients with traumatic brain injury

Visfatin is a newly identified pro-inflammatory adipokine and a genetic polymorphism −1535 C > T located in the visfatin gene promoter has been suggested to be associated with the regulation of visfatin expression in some inflammatory illness. However, there were some conflicting results regarding whether this variant is functional or not. This study aimed to examine the relations of the −1535 C > T single nucleotide polymorphism (SNP) of visfatin gene to the plasma visfatin and C-reactive protein concentrations in traumatic brain injury (TBI). 318 Chinese Han patients with TBI were recruited in this study. Plasma visfatin and C-reactive protein levels were significantly different between the genotypes in the SNP-1535 C > T even after adjustment for age, sex and body mass index. The genotype C–C had the highest plasma visfatin and C-reactive protein concentrations. The plasma visfatin and C-reactive protein concentrations between the variant genotypes C–T and T–T did not differ significantly. Plasma visfatin level was significantly associated with plasma C-reactive protein level using multivariate linear regression. Thus, the SNP-1535 C > T of visfatin gene seemed to be potentially involved in the inflammatory component of TBI through a decreased production of visfatin.     

Dialysis-Associated Hypertension Treated with Telmisartan – DiaTel: A Pilot,Placebo-Controlled,Cross-Over,Randomized Trial

Treatment of hypertension in hemodialysis (HD) patients is characterised by lack of evidence for both the blood pressure (BP) target goal and the recommended drug class to use. Telmisartan, an Angiotensin receptor blocker (ARB) that is metabolised in the liver and not excreted via HD extracorporeal circuit might be particularly suitable for HD patients. We designed and conducted a randomised, placebo-controlled, double-blind and cross-over trial for treatment of dialysis–associated hypertension with telmisartan 80 mg once daily or placebo on top of standard antihypertensive treatment excluding other Renin-Angiotensin-System (RAS) blockers. In 29 patients after randomization we analysed BP after a treatment period of 8 weeks, while 13 started with telmisartan and 16 with placebo; after 8 weeks 11 continued with telmisartan and 12 with placebo after cross-over, respectively. Patients exhibited a significant reduction of systolic pre-HD BP from 141.9±21.8 before to 131.3±17.3 mmHg after the first treatment period with telmisartan or placebo. However, no average significant influence of telmisartan was observed compared to placebo. The latter may be due to a large inter-individual variability of BP responses reaching from a 40 mmHg decrease under placebo to 40 mmHg increase under telmisartan. Antihypertensive co-medication was changed for clinical reasons in 7 out of 21 patients with no significant difference between telmisartan and placebo groups. Our starting hypothesis, that telmisartan on top of standard therapy lowers systolic office BP in HD patients could not be confirmed. In conclusion, this small trial indicates that testing antihypertensive drug efficacy in HD patients is challenging due to complicated standardization of concomitant medication and other confounding factors, e.g. volume status, salt load and neurohormonal activation, that influence BP control in HD patients.

Trial Registration

Clinicaltrialsregister.eu 2005-005021-60     

Periodicity,persistence, and collapse in host–parasitoid systems with egg limitation

There is an emerging consensus that parasitoids are limited by the number of eggs which they can lay as well as the amount of time they can search for their hosts. Since egg limitation tends to destabilize host–parasitoid dynamics, successful control of insect pests by parasitoids requires additional stabilizing mechanisms such as heterogeneity in the distribution of parasitoid attacks and host density-dependence. To better understand how egg limitation, search limitation, heterogeneity in parasitoid attacks, and host density-dependence influence host–parasitoid dynamics, discrete time models accounting for these factors are analyzed. When parasitoids are purely egg-limited, a complete anaylsis of the host–parasitoid dynamics are possible. The analysis implies that the parasitoid can invade the host system only if the parasitoid’s intrinsic fitness exceeds the host’s intrinsic fitness. When the parasitoid can invade, there is a critical threshold, CV ^*>1, of the coefficient of variation (CV) of the distribution of parasitoid attacks that determines that outcome of the invasion. If parasitoid attacks sufficiently aggregated (i.e., CV>CV ^*), then the host and parasitoid coexist. Typically (in a topological sense), this coexistence is shown to occur about a periodic attractor or a stable equilibrium. If the parasitoid attacks are sufficiently random (i.e. CV<CV ^*), then the parasitoid drives the host to extinction. When parasitoids are weakly search-limited as well as egg-limited, coexistence about a global attractor occurs even if CV<CV ^*. However, numerical simulations suggest that the nature of this attractor depends critically on whether CV<1 or CV>1. When CV<1, the parasitoid exhibits highly oscillatory dynamics. Alternatively, when parasitoid attacks are sufficiently aggregated but not overly aggregated (i.e. CV>1 but close to 1), the host and parasitoid coexist about a stable equilibrium with low host densities. The implications of these results for classical biological control are discussed.     

Comparative Prevalence of Immune Evasion Complex Genes Associated with β-Hemolysin Converting Bacteriophages in MRSA ST5 Isolates from Swine,Swine Facilities,Humans with Swine Contact,and Humans with No Swine Contact

Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage’s absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a reduced capacity to cause severe disease in immunocompetent humans.     

Interaction of photons with molecules – cross-sections for photoabsorption, photoionization, and photodissociation

A survey is given of recent progress in measurements of photoabsorption, photoionization, and photodissociation cross-sections of molecules in the wavelength range of photons in the vacuum ultraviolet (VUV), where the optical oscillator-strengths of most molecules are predominantly distributed. Some remarks are presented on molecules in the condensed phase. Particular emphasis is placed on the current understanding of spectroscopy and dissociation dynamics of molecules in the superexcited states which are produced through the interaction of photons in this wavelength range. In the VUV range, most of the observed superexcited states are assigned to high-Rydberg states which are vibrationally (and/or rotationally), doubly, or inner-core excited, and converge to each of the ion states. Received: 2 September 1998 / Accepted in revised form: 10 September 1999     

A phenotypic male with karyotype 45,X/45,X,ace+(?Yq-)

Summary A phenotypical normal 22-year-old male with the sex chromosome constitution 45,X/45,X,ace+(?Yq-) and an atypical endogenous depression has been studied. The chromosome aberration and the depression are most probably not aetiologically connected.The patient presented no physical signs of male Turner phenotype, except for short stature. His personality development was, however, in several ways similar to what is characteristic for females with Turner's syndrome and karyotype 45,X and he had some dermatoglyphic signs similar to females with Turner's syndrome.The cytogenetic findings in leucocytes as well as fibroblast cultures indicated that the small acentric chromosome fragment found in approximately half of the cells was made up of the short arms of a Y chromosome. The finding of only short arms Y chromosome in approximately half of the cells in a phenotypically normal male with testes of normal size supports indications from previous studies that the genes necessary for the development of testes are located in the short arms Y. The finding further indicates that if homologous gene loci for the short arms X are present in the Y chromosome, they must be located in the short arms, and deletion long arms Y is most probably not an aetiological factor in the development of the so-called male Turner phenotype.

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Zusammenfassung Es wird ein phänotypisch normaler 22jähriger Mann mit der Geschlechtschromosomenkonstitution 45,X/45,X,ace+(?Yq-) und einer atypischen endogenen Depression beschrieben. Die Chromosomenaberration und die Depression stehen sehr wahrscheinlich in keinem ätiologischen Zusammenhang.Der Patient zeigte keine körperlichen Veränderungen im Sinne der männlichen Turner-Phänotype, mit Ausnahme von Kleinwuchs. Seine Persönlichkeitsentwicklung jedoch ließ sich in mehreren Punkten mit den Charakteristika, wie man sie bei Frauen mit Turner-Syndrom und der Karyotype 45,X sieht, vergleichen; und er hatte einige dermatoglyphische Zeichen, ähnlich wie bei Frauen mit Turner-Syndrom.Die cytogenetischen Untersuchungsergebnisse sowohl an Leukocyten als auch an Fibroblastenkulturen deuteten an, daß das kleine azentrische Chromosomenfragment, welches sich in ungefähr der Hälfte der Zellen fand, von den kurzen Armen des Y-Chromosoms gebildet wurde. Der Fund von lediglich den kurzen Armen des Y-Chromosoms in ungefähr der Hälfte der Zellen bei einem phänotypisch normalen Mann mit normaler Testisgröße unterstützt die Vermutungen vorangegangener Untersuchungen, daß die Gene, die für die Entwicklung der Testis notwendig sind, auf den kurzen Armen des Y-Chromosoms lokalisiert sind. Das Ergebnis der Untersuchung deutet weiterhin an, wenn homologe Genloci für die kurzen Arme X im Y-Chromosom vorhanden sind, diese auf den kurzen Armen lokalisiert sein müssen und daß eine Deletion der langen Arme des Y-Chromosoms sehr wahrscheinlich kein ätiologischer Faktor in der Entwicklung der sogenannten männlichen Turner-Phänotype ist.

     

Determination of meso-α, ε-Diaminopimelate with meso-α, ε-Diaminopimelate d-Dehydrogenase

A simple and sensitive spectrophotometric method for determining meso-α,ε-diaminopime-late with meso-α,ε-diaminopimelate d-dehydrogenase (EC class 1.4.1) is described. meso-α,ε-Diaminopimelate was determined spectrophotometrically with the enzyme by measuring the NADPH formed (Procedure A) or the formazan produced by NADPH (Procedure B). A linear relationship was established between absorbance and the amount of amino acid (0.02-0.20 μmol). This method can be used to assay diaminopimelate epimerase (EC 5.1.1.7) and is applicable for determining meso-α,ε-diaminopimelate specifically in hydrolyzates of bacterial cell wall.     

Binding of Nonnatural α,β-Oligocytidylates with DNA Duplexes

Binding of short fluorescently labeled AT-containing DNA duplexes with modified oligocytidylates is studied. The latter are modified to contain nonnatural -anomers along with natural -nucleotides; the nucleotide composition is selected according to the putative scheme of noncanonical triplex formation between duplex and oligomer bases. Nondenaturing gel electrophoresis is used to study the interaction of fluorescent duplexes with cytidyl oligomers and oligocytidylate self-association at low temperatures. A DNA duplex of random AT composition is shown to bind with an excess of the corresponding oligocytidylate in 0.1 M Tris-HCl in the presence of Mg²⁺. Binding is observed at neutral pH values, while more basic pH (8.0) prevents binding of the AT duplex and oligocytidylate. Unlike oligonucleotides of random composition, a regular dA₃₀:dT₃₀ duplex does not bind with the dC strand. It is also shown that an alternating self-complementary duplex d(AT)₁₆ and oligocytidylate d(CC)₁₅ do not form complexes, and poly-dC self-associates are formed instead. The effect of 2-O-methylation of the third strand on complex formation and self-association is also analyzed. The results suggest that a modified oligocytidylate binds with a random-composition duplex, albeit with lower efficiency.     

Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides

Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine.