Entrainment of split circadian activity rhythms in hamsters

Hamsters that showed splitting of their circadian rhythms of wheel-running activity following long-term exposure to constant illumination (LL) were exposed to light-dark (LD) cycles with 2-hr dark segments, and with periods of 24.00, 24.23 or 24.72 hr. For comparison, hamsters showing nonsplit rhythms were also studied. In all cases of split rhythms, at least one of the two split components entrained to the LD cycles. In some animals, the second component continued to free-run until it merged with the entrained component, while in others, the second component also entrained to the LD cycle but maintained a stable phase angle of 6-14.5 hr relative to dark onset. These results were obtained in cases where the period of the LD cycle was shorter than that of the split rhythms and in cases where it was longer, implying that split components can be phase-advanced as well as phase-delayed by 2 hr of darkness. Three hamsters that showed stable entrainment of split rhythms were allowed to free-run in LL. The LD cycles were then reinstated, but instead of overlapping with the first component, as it did before, the dark segment was timed to overlap with the second. The entrainment patterns that ensued were similar to the ones obtained during the first LD exposure, indicating that the two split components respond to darkness in a qualitatively similar fashion. These results are further evidence that the pacemaker system underlying split circadian activity rhythms in hamsters is composed of two mutually coupled populations of oscillators that have similar properties, including a bidirectional phase response curve. Such a dual-oscillator organization may also underlie normal, or nonsplit, activity rhythms, as suggested by Pittendrigh and Daan (1976c), but the data are also compatible with the alternative view that the circadian pacemaker consists of a large number of coupled oscillators, which only dissociate into two separate populations in some animals under conditions of moderate LL intensity.     

Abundant DNase I-Sensitive Bacterial DNA in Healthy Porcine Lungs and Its Implications for the Lung Microbiome

Human lungs are constantly exposed to bacteria in the environment, yet the prevailing dogma is that healthy lungs are sterile. DNA sequencing-based studies of pulmonary bacterial diversity challenge this notion. However, DNA-based microbial analysis currently fails to distinguish between DNA from live bacteria and that from bacteria that have been killed by lung immune mechanisms, potentially causing overestimation of bacterial abundance and diversity. We investigated whether bacterial DNA recovered from lungs represents live or dead bacteria in bronchoalveolar lavage (BAL) fluid and lung samples in young healthy pigs. Live bacterial DNA was DNase I resistant and became DNase I sensitive upon human antimicrobial-mediated killing in vitro. We determined live and total bacterial DNA loads in porcine BAL fluid and lung tissue by comparing DNase I-treated versus untreated samples. In contrast to the case for BAL fluid, we were unable to culture bacteria from most lung homogenates. Surprisingly, total bacterial DNA was abundant in both BAL fluid and lung homogenates. In BAL fluid, 63% was DNase I sensitive. In 6 out of 11 lung homogenates, all bacterial DNA was DNase I sensitive, suggesting a predominance of dead bacteria; in the remaining homogenates, 94% was DNase I sensitive, and bacterial diversity determined by 16S rRNA gene sequencing was similar in DNase I-treated and untreated samples. Healthy pig lungs are mostly sterile yet contain abundant DNase I-sensitive DNA from inhaled and aspirated bacteria killed by pulmonary host defense mechanisms. This approach and conceptual framework will improve analysis of the lung microbiome in disease.     

Construction and characterisation of a BAC library for genome analysis of the allotetraploid coffee species (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.Communicated by J.W. Snape     

Chronic total occlusion due to diffuse in-stent restenosis: is brachytherapy the solution?

Percutaneous coronary intervention of chronic total occlusions (CTO) is associated with a significantly higher incidence of reocclusion and restenosis compared with non-total occlusions. Randomized and observational trials have demonstrated the effectiveness of intracoronary brachytherapy (ICBT) for the prevention of recurrent in-stent restenosis. However, limited data are available on the effectiveness of ICBT in patients with totally occluded in-stent restenosis. The authors assessed the long-term outcome of patients treated with intracoronary gamma radiation for totally occluded in-stent restenotic lesions. Percutaneous coronary intervention and subsequent catheter-based irradiation with iridium-192 was performed in 100 patients (103 vessels) with diffuse in-stent restenosis. At baseline, CTO of the target vessel at the site of the stent was present in 15 vessels (14.5%). Follow-up data were collected during follow-up visits and from telephone interviews. Repeat coronary angiography was performed in symptomatic patients with clinical restenosis. Clinical and angiographic characteristics were similar between the two groups, although there was a trend towards more unstable angina at the index procedure in CTO patients (66.7% versus 41.4%; p = 0.12) compared with patients without non-total occlusions. A higher percentage of patients (53.3%) with CTO required longer radiation sources (14 seeds, covering a length of 55 mm), compared with 23.9% of patients with non-total occlusion (p = 0.04). With a mean follow-up period of 47.5 +/- 24.0 months, major adverse cardiac events (MACE) were observed in 10 of 15 patients (66.7%) with CTO compared with 25 out of 88 patients (28.4%) without CTO (p = 0.009). According to multivariate analysis, total occlusion of the target vessel at baseline was the single independent predictor of MACE at one-year follow-up (relative risk 16.2, 95% confidence interval 4.2-62.9; p < 0.0001). This study shows that the use of gamma radiation for the prevention of recurrence of in-stent restenosis in patients with CTO does not seem to be as effective as in patients with non-total occlusions. Furthermore, CTO was an independent predictor of worse outcome at long-term follow-up in this study.     

Linkage disequilibrium and founder effect analysis of the NF1 gene in French Canadians from the Quebec population

We genotyped 19 neurofibromatosis type 1 (NF1) families from French Canadians of the Quebec population with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Linkage analysis of the four microsatellite markers among the 19 NF1 families indicates that the four microsatellites are strongly linked with NF1 disease (LOD = 2.76-3.64). The four markers are associated (P = 0-0.077) except marker pair IVS26-2.3/IVS27AC33.1 (P = 0.18 or 0.17). However, perhaps due to the high mutation rate of the NF1 gene, no founder effect for NF1 was detected in the Quebec French Canadians.     

Synthesis and antimicrobial activity of 2-fluorophenyl-4,6-disubstituted [1,3,5]triazines

A series of 2-fluorophenyl-4,6-disubstituted [1,3,5]triazines (1) and (2) were synthesized and evaluated for their antimicrobial activity against three representative gram-positive bacteria and two fungi. The structure–activity relationship (SAR) demonstrates that the 3- or 4-fluorophenyl component attached directly to the triazine ring was essential for activity. Of these compounds, 14, 15, and 25 demonstrated significant activity against all selected organisms compared to control. These compounds were generally nontoxic and may prove useful as antimicrobial agents.     

Certolizumab pegol plus methotrexate 5-year results from the rheumatoid arthritis prevention of structural damage (RAPID) 2 randomized controlled trial and long-term extension in rheumatoid arthritis patients

IntroductionAs patients with rheumatoid arthritis (RA) receive treatment with anti-tumour necrosis factors over several years, it is important to evaluate their long-term safety and efficacy. The objective of this study was to examine the safety and benefits of certolizumab pegol (CZP)+methotrexate (MTX) treatment for almost 5 years in patients with RA.MethodsPatients who completed the 24-week Rheumatoid Arthritis Prevention of Structural Damage (RAPID) 2 randomized controlled trial (RCT; {"type":"clinical-trial","attrs":{"text":"NCT00160602","term_id":"NCT00160602"}}NCT00160602), or who were American College of Rheumatology (ACR) 20 non-responders at Week 16, entered the open-label extension (OLE; {"type":"clinical-trial","attrs":{"text":"NCT00160641","term_id":"NCT00160641"}}NCT00160641). After ≥6 months treatment with CZP 400 mg every two weeks (Q2W), dose was reduced to 200 mg Q2W, the approved maintenance dose. Safety data are presented from all patients who received ≥1 dose CZP (Safety population, n=612). Efficacy data are presented to Week 232 for the intent-to-treat (ITT, n=492) and Week 24 CZP RCT Completer (n=342) populations, and through 192 weeks of dose-reduction for the Dose-reduction population (patients whose CZP dose was reduced to 200 mg, n=369). Radiographic progression (modified total Sharp score change from RCT baseline >0.5) to Week 128 is reported for the Week 24 CZP Completers.ResultsIn the RCT, 619 patients were randomized to CZP+MTX (n=492) or placebo+MTX (n=127). Overall, 567 patients (91.6%) entered the OLE: 447 CZP and 120 placebo patients. Of all randomized patients, 358 (57.8%) were ongoing at Week 232. Annual drop-out rates during the first four years ranged from 8.4–15.0%. Event rates per 100 patient-years were 163.0 for adverse events (AEs) and 15.7 for serious AEs. Nineteen patients (3.1%) had fatal AEs (incidence rate=0.8). Clinical improvements in the RCT were maintained to Week 232 in the CZP Completers: mean Disease Activity Score 28 (Erythrocyte Sedimentation Rate) change from baseline was −3.4 and ACR20/50/70 responses 68.4%/47.1%/25.1% (non-responder imputation). Similar improvements observed in the ITT were maintained following dose-reduction. 73.2% of CZP Completers had no radiographic progression at Week 128.ConclusionsIn patients with active RA despite MTX therapy, CZP was well tolerated, with no new safety signals identified. CZP provided sustained improvements in clinical outcomes for almost 5 years.

Trial registration

ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT00160602","term_id":"NCT00160602"}}NCT00160602 and {"type":"clinical-trial","attrs":{"text":"NCT00160641","term_id":"NCT00160641"}}NCT00160641. Registered 8 September 2005.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0767-2) contains supplementary material, which is available to authorized users.