Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment

Escherichia coli FtsK is an essential cell division protein, which is thought to pump chromosomal DNA through the closing septum in an oriented manner by following DNA sequence polarity. Here, we perform single-molecule measurements of translocation by FtsK50C, a derivative that functions as a DNA translocase in vitro. FtsK50C translocation follows Michaelis-Menten kinetics, with a maximum speed of approximately 6.7 kbp/s. We present results on the effect of applied force on the speed, distance translocated, and the mean times during and between protein activity. Surprisingly, we observe that FtsK50C can spontaneously reverse its translocation direction on a fragment of E. coli chromosomal DNA, indicating that DNA sequence is not the sole determinant of translocation direction. We conclude that in vivo polarization of FtsK translocation could require the presence of cofactors; alternatively, we propose a model in which tension in the DNA directs FtsK translocation.     

Therapeutic Hypothermia Modifies Perinatal Asphyxia-Induced Changes of the Corpus Callosum and Outcome in Neonates

What Is Known about this Subject?

Diffusion-weighted MRI has demonstrated changes in the corpus callosum of term neonates with perinatal asphyxia. The severity of cerebral changes demonstrated using diffusion-weighted MRI is difficult to assess without measuring values of the Apparent Diffusion Coefficient (ADC).

What Is New?

ADC values of the anterior part of the corpus callosum are slightly higher than of the posterior part in full term infants with perinatal asphyxia. Low ADC values of the corpus callosum were associated with an adverse outcome in infants with perinatal asphyxia. In infants treated with hypothermia lower ADC values than with normothermia were associated with a poor outcome, supporting neuroprotective effects of hypothermia

Background

Using MRI, changes can be detected in the corpus callosum (CC) following perinatal asphyxia which are associated with later neurodevelopmental outcome.

Aim

To study the association between the apparent diffusion coefficient of water (ADC) in the CC on MRI in neonates with perinatal asphyxia and neurodevelopmental outcome at 18 months of age.

Subjects, Methods

Of 121 infants 32 (26%) died and 13 (11%) survived with an adverse neurological outcome. Sixty-five (54%) received therapeutic hypothermia. MRI was performed within 7 days after birth using a 1.5 T or 3.0 T system, and ADC values were measured in the anterior and posterior CC. The association between ADC and composite outcome (death or abnormal neurodevelopment) was analyzed for both normothermia and hypothermia cases using receiver operating characteristics.

Results

ADC values of the posterior CC were lower than of the anterior part (mean difference 0.050 x 10^-3 mm²/s, p<0.001). Field strength did not affect ADC values. ADC values of the posterior part of the CC were significantly lower in infants with basal ganglia/thalamus or near total brain injury (p<0.001). Lower ADC values were associated with an adverse outcome, but cut-off levels were lower after hypothermia (1.024 x 10^-3 mm²/s vs 0.969 x 10^-3 mm²/s)

Conclusion

Low ADC values of the posterior part of the corpus callosum are associated with an adverse outcome in term or near term neonates with perinatal asphyxia. Therapeutic hypothermia slightly modifies this association, showing that lower values were needed for an adverse outcome.     

Attainment and characteristics of clinical remission according to the new ACR-EULAR criteria in abatacept-treated patients with early rheumatoid arthritis: new analyses from the Abatacept study to Gauge Remission and joint damage progression in methotrexate (MTX)-naive patients with Early Erosive rheumatoid arthritis (AGREE)

IntroductionThis study evaluated various remission criteria in abatacept plus methotrexate (MTX)-treated patients with early rheumatoid arthritis (RA). We aimed to investigate the time to, and sustainability of, remission, and to evaluate the relationship between remission, function and structure.MethodsPost hoc analyses were performed from the 12-month, double-blind period of the Abatacept study to Gauge Remission and joint damage progression in methotrexate (MTX)-naive patients with Early Erosive rheumatoid arthritis (AGREE) in patients with early RA (≤2 years) and poor prognostic factors, comparing abatacept plus MTX (n = 210) versus MTX alone (n = 209).ResultsAt month 12, Disease Activity Score 28, Simplified Disease Activity Index (SDAI), Clinical Disease Activity Index and Boolean remission rates were, for abatacept plus MTX versus MTX alone: 47.6 % versus 27.3 %, 33.3 % versus 12.4 %, 34.3 % versus 16.3 %, and 23.8 % versus 5.7 %, respectively. Cumulative probability demonstrated higher proportions achieving first remission and first sustained remission for abatacept plus MTX versus MTX alone (e.g., 23.3 % [95 % confidence interval (CI): 17.6, 29.1] vs 12.9 % [8.4, 17.5] for first SDAI remission over 0–6 months). For patients in SDAI remission at month 3, mean Health Assessment Questionnaire-Disability Index at month 12 was 0.20 versus 0.50 for abatacept plus MTX versus MTX alone. Mean changes in radiographic score from baseline to month 12 were minimal for patients in SDAI remission at month 3 in both groups, while less structural damage progression was seen, 0.75 versus 1.35, respectively, for abatacept plus MTX versus MTX alone for patients with moderate/high disease activity at month 3 (adjusted mean treatment difference: −0.60 [95 % CI: −1.11, −0.09; P < 0.05]).ConclusionsHigh proportions of abatacept plus MTX-treated patients achieved stringent remission criteria. Remission was associated with long-term functional benefit; dissociation was seen between clinical and structural outcomes for abatacept. These findings highlight the impact of reaching stringent remission targets early, on physical function and structural damage, in MTX-naïve biologic-treated patients.

Trial registration

ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00122382","term_id":"NCT00122382"}}NCT00122382. Registered 19 July 2005.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0671-9) contains supplementary material, which is available to authorized users.     

Combining Organophosphate Treated Wall Linings and Long-lasting Insecticidal Nets for Improved Control of Pyrethroid Resistant Anopheles gambiae

Background

New approaches to delivering insecticides need to be developed to improve malaria vector control. Insecticidal durable wall lining (DL) and net wall hangings (NWH) are novel alternatives to indoor residual spraying which can be produced in a long-lasting format. Non-pyrethroid versions could be used in combination with long-lasting insecticidal nets for improved control and management of insecticide resistant vector populations.

Methods

Experimental hut trials were carried out in Valley du Kou, Burkina Faso to evaluate the efficacy of pirimiphos methyl treated DL and NWH either alone or in combination with LLINs against pyrethroid resistant Anopheles gambiae ss. Comparison was made with pyrethroid DL. Mosquitoes were genotyped for kdr and ace-1R resistant genes to investigate the insecticide resistance management potential of the combination.

Results

The overall kdr and ace-1^R allele frequencies were 0.95 and 0.01 respectively. Mortality with p-methyl DL and NWH alone was higher than with pyrethroid DL alone (>95% vs 40%; P<0.001). Combining pyrethroid DL with LLINs did not show improvement in mortality (48%) compared to the LLIN alone (44%) (P>0.1). Combining p-methyl DL or NWH with LLINs reduced biting rates significantly (8–9%) compared to p-methyl DL and NWH alone (>40%) and killed all An gambiae that entered the huts. Mosquitoes bearing the ace-1^R gene were more likely to survive in huts with p-methyl DL alone (p<0.03) whereas all resistant and susceptible genotypes were killed by the combination.

Conclusion

P-methyl DL and NWH outperformed pyrethroid DL. Combining p-methyl DL and NWH with LLINs could provide significant epidemiological benefits against a vector population which is resistant to pyrethroids but susceptible to organophosphates. There was evidence that the single intervention would select kdr and ace-1^R resistance genes and the combination intervention might select less strongly. Technology to bind organophosphates to plastic wall lining would be worth developing.     

Reshaping of bulbar odor response by nasal flow rate in the rat

Background

The impact of respiratory dynamics on odor response has been poorly studied at the olfactory bulb level. However, it has been shown that sniffing in the behaving rodent is highly dynamic and varies both in frequency and flow rate. Bulbar odor response could vary with these sniffing parameter variations. Consequently, it is necessary to understand how nasal airflow can modify and shape odor response at the olfactory bulb level.

Methodology and Principal Findings

To assess this question, we used a double cannulation and simulated nasal airflow protocol on anesthetized rats to uncouple nasal airflow from animal respiration. Both mitral/tufted cell extracellular unit activity and local field potentials (LFPs) were recorded. We found that airflow changes in the normal range were sufficient to substantially reorganize the response of the olfactory bulb. In particular, cellular odor-evoked activities, LFP oscillations and spike phase-locking to LFPs were strongly modified by nasal flow rate.

Conclusion

Our results indicate the importance of reconsidering the notion of odor coding as odor response at the bulbar level is ceaselessly modified by respiratory dynamics.     

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four C_α-thioether bridges. These bridges, which are essential for RumC''s antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether–containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by C_α H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,β-dehydro-amino acid intermediate during C_α-thioether bridge LC–MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.     

Prevention of Mother-To-Child Transmission of HIV: Cost-Effectiveness of Antiretroviral Regimens and Feeding Options in Rwanda

Background

Rwanda''s National PMTCT program aims to achieve elimination of new HIV infections in children by 2015. In November 2010, Rwanda adopted the WHO 2010 ARV guidelines for PMTCT recommending Option B (HAART) for all HIV-positive pregnant women extended throughout breastfeeding and discontinued (short course-HAART) only for those not eligible for life treatment. The current study aims to assess the cost-effectiveness of this policy choice.

Methods

Based on a cohort of HIV-infected pregnant women in Rwanda, we modelled the cost-effectiveness of six regimens: dual ARV prophylaxis with either 12 months breastfeeding or replacement feeding; short course HAART (Sc-HAART) prophylaxis with either 6 months breastfeeding, 12 months breastfeeding, or 18 months breastfeeding; and Sc-HAART prophylaxis with replacement feeding. Direct costs were modelled based on all inputs in each scenario and related unit costs. Effectiveness was evaluated by measuring HIV-free survival at 18 months. Savings correspond to the lifetime costs of HIV treatment and care avoided as a result of all vertical HIV infections averted.

Results

All PMTCT scenarios considered are cost saving compared to “no intervention.” Sc-HAART with 12 months breastfeeding or 6 months breastfeeding dominate all other scenarios. Sc-HAART with 12 months breastfeeding allows for more children to be alive and HIV-uninfected by 18 months than Sc-HAART with 6 months breastfeeding for an incremental cost per child alive and uninfected of 11,882 USD. This conclusion is sensitive to changes in the relative risk of mortality by 18 months for exposed HIV-uninfected children on replacement feeding from birth and those who were breastfed for only 6 months compared to those breastfeeding for 12 months or more.

Conclusion

Our findings support the earlier decision by Rwanda to adopt WHO Option B and could inform alternatives for breastfeeding duration. Local contexts and existing care delivery models should be part of national policy decisions.     

Evolution of oil-producing trichomes in Sisyrinchium (Iridaceae): insights from the first comprehensive phylogenetic analysis of the genus

Background and Aims

Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined.

Methods

Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species.

Key Results and Conclusions

Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.     

Translation of HLA-HIV associations to the cellular level: HIV adapts to inflate CD8 T cell responses against Nef and HLA-adapted variant epitopes

Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-γ responses in 290 individuals from the same cohort, which gave rise to 874 HLA-HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-γ responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA-HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for diversity coverage approaches in HIV vaccine design.