The ECHELON-2 Trial: 5-year results of a randomized, phase III study of brentuximab vedotin with chemotherapy for CD30-positive peripheral T-cell lymphoma.

BACKGROUND: For patients with peripheral T-cell lymphoma (PTCL), outcomes using frontline treatment with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like therapy are typically poor. The ECHELON-2 study demonstrated that brentuximab vedotin plus cyclophosphamide, doxorubicin, and prednisone (A+CHP) exhibited statistically superior progression-free survival (PFS) per independent central review and improvements in overall survival versus CHOP for the frontline treatment of patients with systemic anaplastic large cell lymphoma or other CD30-positive PTCL. PATIENTS AND METHODS: ECHELON-2 is a double-blind, double-dummy, randomized, placebo-controlled, active-comparator phase III study. We present an exploratory update of the ECHELON-2 study, including an analysis of 5-year PFS per investigator in the intent-to-treat analysis group. RESULTS: A total of 452 patients were randomized (1 : 1) to six or eight cycles of A+CHP (N = 226) or CHOP (N = 226). At median follow-up of 47.6 months, 5-year PFS rates were 51.4% [95% confidence interval (CI): 42.8% to 59.4%] with A+CHP versus 43.0% (95% CI: 35.8% to 50.0%) with CHOP (hazard ratio = 0.70; 95% CI: 0.53-0.91), and 5-year overall survival (OS) rates were 70.1% (95% CI: 63.3% to 75.9%) with A+CHP versus 61.0% (95% CI: 54.0% to 67.3%) with CHOP (hazard ratio = 0.72; 95% CI: 0.53-0.99). Both PFS and OS were generally consistent across key subgroups. Peripheral neuropathy was resolved or improved in 72% (84/117) of patients in the A+CHP arm and 78% (97/124) in the CHOP arm. Among patients who relapsed and subsequently received brentuximab vedotin, the objective response rate was 59% with brentuximab vedotin retreatment after A+CHP and 50% with subsequent brentuximab vedotin after CHOP. CONCLUSIONS: In this 5-year update of ECHELON-2, frontline treatment of patients with PTCL with A+CHP continues to provide clinically meaningful improvement in PFS and OS versus CHOP, with a manageable safety profile, including continued resolution or improvement of peripheral neuropathy.

Incidence and prevalence of rheumatoid arthritis in Denmark from 1998 to 2018: a nationwide register-based study.

OBJECTIVE: To investigate the incidence and prevalence of rheumatoid arthritis (RA) in the adult Danish population. METHOD: In this nationwide register-based cohort study, patients with incident RA between 1998 and the end of 2018 were identified using Danish administrative registries. The age- and sex-standardized incidence rate (IR), incidence proportion (IP), lifetime risk (LR), and point prevalence (PP) of RA were calculated. RA was defined as a first-time RA diagnosis registered in the Danish National Patient Registry combined with a redeemed prescription of a conventional synthetic disease-modifying anti-rheumatic drug in the following year. In addition, three different case definitions of RA were explored. RESULTS: The overall age- and sex-standardized IR of RA from 1998 to 2018 was 35.5 [95% confidence interval (CI) 35.1-35.9] per 100 000 person-years while the IP was 35.2 (95% CI 34.8-35.5) per 100 000 individuals. The IR was two-fold higher for women than for men. The LR of RA ranged from 2.3% to 3.4% for women and from 1.1% to 1.5% for men, depending on the RA case definition used. The overall PP of RA was 0.6% (95% CI 0.5-0.6%) in 2018: 0.8% (95% CI 0.7-0.8%) for women and 0.3% (95% CI 0.3-0.4%) for men. The prevalence increased about 1.5-fold from 2000 to 2018. CONCLUSION: The IR and PP were approximately two-fold higher for women than for men. The prevalence of RA in Denmark increased significantly from 2000 to 2018. The RA case definition had more impact on the results than the choice of denominator.

Beneficial effect of n-3 polyunsaturated fatty acids on inflammation and analgesic use in psoriatic arthritis: a randomized, double blind, placebo-controlled trial.

OBJECTIVE: This study aimed to investigate the effects of marine n-3 polyunsaturated fatty acids (PUFAs) on disease activity, use of analgesics, and inflammatory biomarkers in patients with psoriatic arthritis (PsA). METHOD: Patients with established PsA (n = 145) were investigated in a randomized, double-blind, placebo-controlled study. The participants received a supplement of 3 g n-3 PUFA/day or 3 g olive oil/day (control) for 24 weeks. Outcome measures for disease activity, use of analgesics, and leukotriene formation from activated granulocytes were assessed at baseline and at study end. RESULTS: In total, 145 patients were included and 133 completed the study. After 24 weeks, the n-3 PUFA group showed a decrease in Disease Activity Score (DAS28-CRP), 68 tender joint count, enthesitis score, and psoriasis area and severity index, although not significantly different from the controls. There was a significant reduction in non-steroidal anti-inflammatory drug (NSAID) and paracetamol use compared with controls (p = 0.04). In addition, the participants in the n-3 PUFA group had significantly lower formation of leukotriene B4 (p = 0.004) from stimulated granulocytes and significantly higher formation of leukotriene B5 (p < 0.001) compared with controls. CONCLUSION: The n-3 PUFA-supplemented group showed improvement in outcome measures for disease activity, although the difference between the groups was not statistically significant. However, use of NSAIDs and paracetamol was significantly reduced in the n-3 PUFA group compared to the control group. Finally, there was a significant decrease in leukotriene B4 formation in the n-3 PUFA group compared with controls.

A novel deletion partly removing the AVP gene causes autosomal recessive inheritance of early-onset neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus (FNDI) typically presents with age-dependent penetrance and autosomal dominant inheritance caused by missense variations in one allele of the AVP gene encoding the arginine vasopressin (AVP) prohormone. We present the molecular genetic characteristics underlying an unusual form of FNDI occurring with very early onset and seemingly autosomal recessive inheritance. By DNA amplification and sequencing, we identified a novel variant allele of the AVP gene carrying a 10,396 base pair deletion involving the majority of the AVP gene as well as its regulatory sequences in the intergenic region between the AVP and the OXT gene, encoding the oxytocin prohormone. We found two chromosomes carrying the deletion in affected family members and one in unaffected family members suspected to transmit the deleted allele. Whole-genome array analysis confirmed the results and excluded the presence of any additional major pathogenic abnormalities. The deletion is predicted to abolish the transcription of the AVP gene, thus the fact that family members heterozygous for the deletion remain healthy argues, in general, against haploinsufficiency as the pathogenic mechanism FNDI. Accordingly, our data is strong support to the prevailing idea that dominant inheritance of FNDI is due to a dominant-negative effect exerted by variant AVP prohormone.

Fluorochemicals used in food packaging inhibit male sex hormone synthesis.

Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS. The aim of this study was to elucidate the effects of six fluorochemicals on sex hormone synthesis and androgen receptor (AR) activation in vitro. Four PAPS and two metabolites, perfluorooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (8:2 FTOH) were tested. Hormone profiles, including eight steroid hormones, generally showed that 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH led to decreases in androgens (testosterone, dehydroepiandrosterone, and androstenedione) in the H295R steroidogenesis assay. Decreases were observed for progesterone and 17-OH-progesterone as well. These observations indicated that a step prior to progestagen and androgen synthesis had been affected. Gene expression analysis of StAR, Bzrp, CYP11A, CYP17, CYP21 and CYP19 mRNA showed a decrease in Bzrp mRNA levels for 8:2 monoPAPS and 8:2 FTOH indicating interference with cholesterol transport to the inner mitochondria. Cortisol, estrone and 17ß-estradiol levels were in several cases increased with exposure. In accordance with these data CYP19 gene expression increased with 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH exposures indicating that this is a contributing factor to the decreased androgen and the increased estrogen levels. Overall, these results demonstrate that fluorochemicals present in food packaging materials and their metabolites can affect steroidogenesis through decreased Bzrp and increased CYP19 gene expression leading to lower androgen and higher estrogen levels.

Cardiac autonomic dysfunction in hemodialysis patients assessed by heart rate variability.

Sudden cardiac death (SCD) is a major problem in patients with end stage renal disease (ESRD) on haemodialysis (HD) and several risk factors are recognized. A major problem may be the autonomic dysfunction which is observed in more than 50 % of ESRD patients. Autonomic dysfunction, and in particular cardiac autonomic dysfunction, is associated with a high risk of ventricular arrhythmias which may eventually lead to SCD. A non-invasive method for measuring cardiac autonomic function is by assessing heart rate variability (HRV) and this review will focus on HRV in HD patients. ESRD patients have an impaired HRV which may partly explain their high risk of SCD. Some intervention studies in HD patients using HRV as a surrogate parameter have been published and are also discussed in this review.

NT-proBNP and stem cell factor plasma concentrations are independently associated with cardiovascular outcomes in end-stage renal disease hemodialysis patients.

Aims: End-stage renal disease (ESRD) treated by chronic hemodialysis (HD) is associated with poor cardiovascular (CV) outcomes, with no available evidence-based therapeutics. A multiplexed proteomic approach may identify new pathophysiological pathways associated with CV outcomes, potentially actionable for precision medicine. Methods and results: The AURORA trial was an international, multicentre, randomized, double-blind trial involving 2776 patients undergoing maintenance HD. Rosuvastatin vs. placebo had no significant effect on the composite primary endpoint of death from CV causes, nonfatal myocardial infarction or nonfatal stroke. We first compared CV risk-matched cases and controls (n = 410) to identify novel biomarkers using a multiplex proximity extension immunoassay (276 proteomic biomarkers assessed with OlinkTM). We replicated our findings in 200 unmatched cases and 200 controls. External validation was conducted from a multicentre real-life Danish cohort [Aarhus-Aalborg (AA), n = 331 patients] in which 92 OlinkTM biomarkers were assessed. In AURORA, only N-terminal pro-brain natriuretic peptide (NT-proBNP, positive association) and stem cell factor (SCF) (negative association) were found consistently associated with the trial's primary outcome across exploration and replication phases, independently from the baseline characteristics. Stem cell factor displayed a lower added predictive ability compared with NT-ProBNP. In the AA cohort, in multivariable analyses, BNP was found significantly associated with major CV events, while higher SCF was associated with less frequent CV deaths. Conclusions: Our findings suggest that NT-proBNP and SCF may help identify ESRD patients with respectively high and low CV risk, beyond classical clinical predictors and also point at novel pathways for prevention and treatment.

Blending of heritable recognition cues among ant nestmates creates distinct colony gestalt odours but prevents within-colony nepotism.

The evolution of sociality is facilitated by the recognition of close kin, but if kin recognition is too accurate, nepotistic behaviour within societies can dissolve social cohesion. In social insects, cuticular hydrocarbons act as nestmate recognition cues and are usually mixed among colony members to create a Gestalt odour. Although earlier studies have established that hydrocarbon profiles are influenced by heritable factors, transfer among nestmates and additional environmental factors, no studies have quantified these relative contributions for separate compounds. Here, we use the ant Formica rufibarbis in a cross-fostering design to test the degree to which hydrocarbons are heritably synthesized by young workers and transferred by their foster workers. Bioassays show that nestmate recognition has a significant heritable component. Multivariate quantitative analyses based on 38 hydrocarbons reveal that a subset of branched alkanes are heritably synthesized, but that these are also extensively transferred among nestmates. In contrast, especially linear alkanes are less heritable and little transferred; these are therefore unlikely to act as cues that allow within-colony nepotistic discrimination or as nestmate recognition cues. These results indicate that heritable compounds are suitable for establishing a genetic Gestalt for efficient nestmate recognition, but that recognition cues within colonies are insufficiently distinct to allow nepotistic kin discrimination.

The effect of N-3 fatty acids on plasma myeloperoxidase levels in healthy adults.

OBJECTIVE: To examine the effect of marine n-3 polyunsaturated fatty acids (PUFA) supplementation in a low to moderate and a high dose on plasma levels of myeloperoxidase (MPO) in healthy individuals. BACKGROUND: Atherosclerosis is a chronic inflammatory disease and MPO, which is secreted primarily from activated neutrophils and monocytes, has pro-inflammatory properties and has been linked with both initiation and propagation of atherosclerosis. Marine n-3 PUFA have anti-inflammatory properties, but whether n-3 PUFA affect plasma levels of MPO is largely unknown. METHODS: Sixty healthy adults were randomized to three groups receiving either 6.6 g PUFA/day, 2.0 g PUFA/day or a control oil (olive oil) for 12 weeks. Blood samples were drawn at baseline and after exposure. Plasma levels of MPO were measured using a MPO ELISA-kit (from Mercodia, Uppsala, Sweden) with specific mouse monoclonal antibodies. RESULTS: Plasma MPO concentrations (microg/L) at baseline were 36.9 +/- 9.4; 36.2 +/- 7.1 and 35.4 +/- 11.3 (for high dose-, low dose- and control-group, respectively). After 12 weeks of supplementation we found no significant changes in plasma MPO in any of the groups nor between groups, with values after intervention of 36.1 +/- 8.6; 37.0 +/- 8.2 and 34.4 +/- 11.1, respectively. CONCLUSION: Supplementation with n-3 PUFA has no effect on plasma levels of MPO in healthy adults with low baseline levels of MPO.

N-3 fatty acids and cardiac autonomic function in humans.

Studies suggest that marine n-3 polyunsaturated fatty acids (PUFA) offer some protection against sudden cardiac death (SCD). The autonomic nervous system is involved in the pathogenesis of SCD and due to the fact that n-3 PUFA is abundant in the brain and other nervous tissue it is likely that n-3 PUFA might modulate autonomic control of the heart. Heart rate variability (HRV) is a non-invasive marker of cardiac autonomic function and an attenuated HRV is a predictor for SCD and arrhythmic events. Studies on HRV and n-3 PUFA have been performed in several populations such as patients with ischaemic heart disease, patients with diabetes mellitus, patients with chronic renal failure, and in healthy subjects. Many studies have demonstrated a positive association between cellular content of n-3 PUFA and HRV as well as supplementation with n-3 PUFA seems to increase HRV and thereby decreasing the risk of arrhythmic events and SCD.

Effects of marine n-3 fatty acids on circulating levels of soluble adhesion molecules in patients with chronic heart failure.

Inflammatory markers as circulating soluble cellular adhesion molecules (sCAMs) and high sensitive C-reactive protein (hsCRP) are elevated in patients with chronic heart failure (CHF), and may constitute an increased risk of adverse outcome. Marine n-3 polyunsaturated fatty acids ( n-3 PUFA) may have anti-inflammatory effect and reduce levels of sCAMs (soluble intercellular adhesion molecule-1 (sICAM-1), vascular adhesion molecule-1 (sVCAM-1), P-selectin) and hsCRP. In a randomized, controlled trial, 138 patients with NYHA class II-III CHF were allocated to receive a daily supplement of 0.9 g of n-3 PUFA or olive oil for 24 weeks. After supplementation, no significant changes occurred in sCAMs or hsCRP after adjusting for possible confounders. However, a significant reduction was observed in sP-selectin in patients receiving n-3 PUFA, but this result was only of borderline significance in a between-group analysis. In conclusion, a daily supplement with 0.9 g of n-3 PUFA does not significantly affect plasma levels of sCAMs or hs-CRP in patients with CHF. n-3 PUFA may reduce sP-selectin, indicating a possible effect on platelet (and endothelial) activation. The results also indicate that the low dose of n-3 PUFA used in many intervention trials does not have deleterious effects on sCAMs or hsCRP.

Serum concentrations of matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1 and alpha2-macroglobulin in healthy subjects after supplementation with different doses of marine n-3 fatty acids.

OBJECTIVE: Extracellular matrix modification by matrix metalloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) and alpha2-macroglobulin may affect the stability of atherosclerotic plaques. Marine n-3 polyunsaturated fatty acids (n-3 PUFA) may protect against plaque rupture. The aim was to investigate the effect of marine n-3 PUFA supplementation on serum levels of MMP-9, TIMP-1, and alpha2-macroglobulin. METHODS: Healthy volunteers were randomized to receive capsules contributing either 6.6 g marine n-3 PUFA/day, 2.0 g marine n-3 PUFA/day or 6.6 g of olive oil (control). Serum MMP-9, TIMP-1 and alpha2-macroglobulin was measured at baseline and after 12 weeks of supplementation. One way ANOVA or Kruskal-Wallis test was used to compare groups. RESULTS: 60 healthy volunteers were enrolled and no subjects dropped out of the 12 week study. There were no statistically significant changes in serum levels of MMP-9, TIMP-1, and alpha2-macroglobulin in any of the three treatment groups (P=0.85, P=0.23 and P=0.87, respectively). CONCLUSION: Supplementation with marine n-3 PUFA had no effect on serum levels of MMP-9, TIMP-1 and alpha2-macroglobulin in healthy subjects. The possible protection offered by marine n-3 PUFA against plaque rupture is therefore unlikely to be mediated through a change in serum levels of MMP-9, TIMP-1 and alpha2-macroglobulin.

NT-pro-BNP is an independent predictor of mortality in patients with end-stage renal disease.

AIM: Patients with end-stage renal disease (ESRD) have an increased mortality from cardiovascular disease (CVD). N-terminal pro-brain natriuretic peptide (NT-pro-BNP) is an independent predictor of mortality in patients with ischemic heart disease and congestive heart failure. Previous data have shown markedly elevated levels of NT-pro-BNP in patients with ESRD, while the prognostic value of elevated levels of NT-pro-BNP in patients with ESRD is largely unknown. The aim of the present study was to examine if the level of NT-pro-BNP predicts mortality in patients with ERSD and CVD. METHODS: We prospectively followed 206 patients with ESRD and documented CVD. Levels of NT-pro-BNP were measured at baseline, and patients were followed for 2 years or until they reached the predefined endpoint of all-cause mortality. RESULTS: During follow-up, the total mortality was 44% (90/206). Patients who died were followed for a median of 314 days (interquartile range 179 - 530). Using Cox regression analysis, age, female sex, systolic blood pressure, dialysis efficiency and plasma levels of NT-pro-BNP were independent prognostic risk factors of mortality. In receiver operating characteristic curve analysis a cut off value for NT-pro-BNP was determined. Patients with values of NT-pro-BNP above 12.200 pg/ml had a 3 times higher risk of death than patients below the cut-off value (HR 3.05 95% CI 1.96 - 4.77, p < 0.0001). CONCLUSION: In spite of generally elevated levels of NT-pro-BNP, NT-pro-BNP is still an independent predictor of mortality and might add prognostic information in patients with ESRD and documented CVD.

Combining electrokinetic transport and bioremediation for enhanced removal of crude oil from contaminated marine sediments: Results of a long-term, mesocosm-scale experiment.

Marine sediments represent an important sink of harmful petroleum hydrocarbons after an accidental oil spill. Electrobioremediation techniques, which combine electrokinetic transport and biodegradation processes, represent an emerging technological platform for a sustainable remediation of contaminated sediments. Here, we describe the results of a long-term mesocosm-scale electrobioremediation experiment for the treatment of marine sediments contaminated by crude oil. A dimensionally stable anode and a stainless-steel mesh cathode were employed to drive seawater electrolysis at a fixed current density of 11 A/m2. This approach allowed establishing conditions conducive to contaminants biodegradation, as confirmed by the enrichment of Alcanivorax borkumensis cells harboring the alkB-gene and other aerobic hydrocarbonoclastic bacteria. Oil chemistry analyses indicated that aromatic hydrocarbons were primarily removed from the sediment via electroosmosis and low molecular weight alkanes (nC6 to nC10) via biodegradation.

Plasma levels of marine n-3 fatty acids and cardiovascular risk markers in renal transplant recipients.

BACKGROUND/OBJECTIVES: Cardiovascular (CV) disease is the leading cause of death after renal transplantation. Marine n-3 polyunsaturated fatty acids (PUFAs) exert potential cardio-protective metabolic effects and might reduce CV morbidity and mortality in renal transplant recipients (RTRs). SUBJECTS/METHODS: In this cross-sectional study of 1990 Norwegian RTRs, transplanted between 1999 and 2011, associations between plasma phospholipid marine n-3 PUFA levels and various CV risk markers at 10 weeks after transplant were evaluated. RESULTS: Higher plasma marine n-3 PUFA levels were associated with lower resting heart rate (rHR), lower fasting plasma glucose (fPG) levels, lower plasma triglyceride levels and higher plasma high-density lipoprotein (HDL) cholesterol levels. Plasma levels of eicosapentaenoic acid, but not docosahexaenoic acid, showed a positive association with plasma HDL cholesterol levels. Plasma marine n-3 PUFA levels were not associated with plasma low-density lipoprotein cholesterol levels, pulse wave velocity or systolic and diastolic blood pressure. A negative association between plasma marine n-3 PUFA levels and CV mortality was weakened by additional adjustment for plasma triglyceride levels and rHR. The ratio of n-6 to n-3 PUFAs showed similar associations with CV risk markers as absolute plasma marine n-3 PUFA levels. CONCLUSIONS: This is the first study in RTRs showing that marine n-3 PUFAs are negatively associated with rHR and fPG in addition to beneficial effects on plasma HDL cholesterol and triglyceride levels. Especially, effects on autonomic nervous function and triglyceride metabolism might contribute to explain the lower CV mortality risk with higher plasma marine n-3 PUFA levels previously shown in this cohort.

Marine n-3 fatty acids, wine intake, and heart rate variability in patients referred for coronary angiography.

BACKGROUND: Dietary n-3 polyunsaturated fatty acids (PUFAs) derived from fish may reduce the incidence of sudden cardiac death (SCD). In addition, wine drinking is suggested to have a protective effect against cardiovascular death. METHODS AND RESULTS: We included 291 patients referred for coronary angiography in whom ischemic heart disease was suspected and all of whom completed a food questionnaire regarding fish and wine intake. The n-3 PUFA composition of granulocyte membranes and of adipose tissue was measured. In addition, 24-hour heart rate variability (HRV) was analyzed. Fish intake was positively associated with the level of n-3 PUFAs in adipose tissue. Significant positive correlation coefficients were found between HRV indices and the levels of n-3 PUFAs in granulocytes. Wine intake was also significantly positively related to HRV, but the patients with the highest wine intake also had the highest intake of fish, as documented by a high n-3 PUFA content in adipose tissue. Multiple linear regression analysis revealed that traditional factors such as treatment with ss-blockers, smoking, age, and previous myocardial infarction were independently related to HRV, and furthermore that n-3 PUFAs (but not wine intake) were significantly independently associated with HRV. CONCLUSIONS: The close positive association between n-3 PUFAs and HRV in patients suspected of having ischemic heart disease may indicate a protective effect of n-3 PUFAs against SCD. This may partly explain the reduction in SCD observed in humans with a modest intake of n-3 PUFA. Wine intake was also positively correlated with HRV, but this correlation was no longer significant after controlling for the cellular level of n-3 PUFA.

Dissection of the DNA-binding domain of Xenopus laevis TFIIIA. Quantitative DNase I footprinting analysis of specific complexes between a 5 S RNA gene fragment and N-terminal fragments of TFIIIA containing three, four or five zinc-finger domains.

Recombinant zinc finger proteins corresponding to N-terminal fragments of Xenopus laevis transcription factor IIIA (TFIIIA) comprising three, four and five fingers produced in Escherichia coli as cleavable hybrid proteins were shown to form specific stoichiometric complexes with DNA fragments containing the internal control region (ICR) of a 5 S RNA gene. The ordered set of DNase I footprints of each of the three proteins on the ICR comprise a nested set of footprints extending upstream from its 3' end (position +96 relative to start of the mature transcript) 20 bp, 20 bp or 34 bp into the ICR, respectively. Quantitative analysis of the footprinting data provided firm evidence that the DNase I footprint, and hence the structure, of the authentic TFIIIA:ICR complex in this region is fully and precisely accounted for by the N-terminal three fingers binding within the +77 to +96 region plus the pair of fingers 4 and 5, both required to extend the footprint upwards from the +77 to the +63 position. A structural interpretation of this set of new footprinting data in view of previous results and data is presented and discussed in terms of a refined model in which the protein-DNA interaction between the ICR and the three N-terminal fingers corresponds closely to that observed in the homologous three-finger zif268:DNA complex, whereas the basic mode of protein-DNA interaction, in which the pair of fingers 4 and 5 is engaged in forming the TFIIIA:ICR complex is of an entirely different, albeit not yet understood nature. To allow assessment of our model in terms of potential specificity-determining H-bonding patterns, a molecular model of the complex between the three-finger TFIIIA fragment and the ICR was constructed, using the zif268:DNA co-ordinates. Eight out of the nine amino acid residues, which according to our model are suitably located for forming hydrogen bonds with the bases, are potential H-bond acceptors or donors.

An untargeted gas chromatography mass spectrometry metabolomics platform for marine polychaetes.

The development of an appropriate extraction method for untargeted environmental metabolomic analysis of marine polychaetes could promote their use for environmental monitoring purposes. To this end, we compared four extraction methods on the marine polychaete Nereis virens both exposed to crude oil and non-exposed. XCMS was used for feature detection and preprocessing; different normalization and scaling approaches were tested; and principal component analysis (PCA) was used together with basic statistical tests to ascertain common metabolic patterns and determine the most suitable extraction method. We conclude that a two-step extraction procedure with 80:20 (v/v) methanol:water on freeze dried polychaete tissue provides the best trade-off between analysis time, and extraction efficiency and intermediate reproducibility. No definitive conclusions could be drawn about the ability of the method to discriminate controls and crude oils in actual biological replicates because the experiment was carried out by design on analytical replicates only. We show that the normalization to the sum of all the common features, and the use of a weighted least squares criterion to fit the PCA by means of scaling to the median absolute deviation (MAD) of the pooled quality control samples significantly improved the clustering of controls and crude oil exposed samples. The scaling alone led to an increase of 19% in explained variance compared to ordinary PCA.

Sequence-specific binding of the N-terminal three-finger fragment of Xenopus transcription factor IIIA to the internal control region of a 5S RNA gene.

An N-terminal fragment of Xenopus TFIIIA, containing domains 1-3, (TF 3), was expressed in E. coli. High yields of recombinant zinc finger protein was isolated, and its DNA binding activity for the internal control region (ICR) of the Xenopus 5S RNA gene, was demonstrated by band-shift experiments and DNase I footprinting analysis. TF 3 protects 20 bp of ICR against DNase I digestion. The limits of protection are from +77 to +96 on both coding and noncoding strand. This protection pattern is identical to the protection pattern obtained with TFIIIA in the overlapping region, showing that the 3-finger fragment accounts fully for the protein-DNA interactions in TFIIIA-5S RNA gene over this region.