Dark states in the light-harvesting complex 2 revealed by two-dimensional electronic spectroscopy

Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity. However the mechanisms behind this process have not yet been fully understood. Recently it was proposed that low-lying energy dark states, such as charge transfer states and polaron pairs, play an important role in the dynamics and directionality of energy transfer. However, it is difficult to directly detect those states because of their small transition dipole moment and overlap with the B850/B870 exciton bands. Here we present a new experimental approach, which combines the selectivity of two-dimensional electronic spectroscopy with the availability of genetically modified light harvesting complexes, to reveal the presence of those dark states in both the genetically modified and the wild-type light harvesting 2 complexes of Rhodopseudomonas palustris. We suggest that Nature has used the unavoidable charge transfer processes that occur when LH pigments are concentrated to enhance and direct the flow of energy

Improved detection of artifactual viral minority variants in high-throughput sequencing data

textabstractHigh-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the Illumina HiSeq2000 library generation and HTS process were investigated by determining minority variant frequencies in an influenza A/WSN/1933(H1N1) virus reverse-genetics plasmid pool. Errors related to amplification and sequencing were determined using the same plasmid pool, by generation of infectious virus using reverse genetics followed by in duplo reverse-transcriptase PCR (RT-PCR) amplification and HTS in the same sequence run. Results showed that after "best practice" quality control (QC), within the plasmid pool, one minority variant with a frequency >0.5% was identified, while 84 and 139 were identified in the RT-PCR amplified samples, indicating RT-PCR amplification artificially increased variation. Detailed analysis showed that artifactual minority variants could be identified by two major technical characteristics: their predominant presence in a single read orientation and uneven distribution of mismatches over the length of the reads. We demonstrate that by addition of two QC steps 95% of the artifactual minority variants could be identified. When our analysis approach was applied to three clinical samples 68% of the initially identified minority variants were identified as artifacts. Our study clearly demonstrated that, without additional QC steps, overestimation of viral minority variants is very likely to occur, mainly as a consequence of the required RT-PCR amplification step. The improved ability to detect and correct for artifactual minority variants, increases data resolution and could aid both past and future studies incorporating HTS. The source code has been made available through Sourceforge (https://sourceforge.net/projects/mva-ngs)

Cardiovascular risk in high-hazard occupations: the role of occupational cardiology

Abstract Work is beneficial for health, but many individuals develop cardiovascular disease (CVD) during their working lives. Occupational cardiology is an emerging field that combines traditional cardiology sub-specialisms with prevention and risk management unique to specific employment characteristics and conditions. In some occupational settings incapacitation through CVD has the potential to be catastrophic due to the nature of work and/or the working environment. These are often termed 'hazardous' or 'high-hazard' occupations. Consequently, many organizations that employ individuals in high-hazard roles undertake pre-employment medicals and periodic medical examinations to screen for CVD. The identification of CVD that exceeds predefined employer (or regulatory body) risk thresholds can result in occupational restriction, or disqualification, which may be temporary or permanent. This article will review the evidence related to occupational cardiology for several high-hazard occupations related to aviation and space, diving, high altitude, emergency workers, commercial transportation, and the military. The article will focus on environmental risk, screening, surveillance, and risk management for the prevention of events precipitated by CVD. Occupational cardiology is a challenging field that requires a broad understanding of general cardiology, environmental, and occupational medicine principles. There is a current lack of consensus and contemporary evidence which requires further research. Provision of evidence-based, but individualized, risk stratification and treatment plans is required from specialists that understand the complex interaction between work and the cardiovascular system. There is a current lack of consensus and contemporary evidence in occupational cardiology and further research is required

Energy Flow in the Cryptophyte PE545 Antenna Is Directed by Bilin Pigment Conformation

Structure-based calculations are combined with quantitative modeling of spectra and energy transfer dynamics to detemine the energy transfer scheme of the PE545 principal light-harvesting antenna of the cryptomonad Rhodomonas CS24. We use a recently developed quantum-mechanics/molecular mechanics (QM/MM) method that allows us to account for pigment-protein interactions at atomic detail in site energies, transition dipole moments, and electronic couplings. In addition, conformational flexibility of the pigment-protein complex is accounted for through molecular dynamics (MD) simulations. We find that conformational disorder largely smoothes the large energetic differences predicted from the crystal structure between the pseudosymmetric pairs PEB50/61C-PEB50/61D and PEB82C-PEB82D. Moreover, we find that, in contrast to chlorophyll-based photosynthetic complexes, pigment composition and conformation play a major role in defining the energy ladder in the PE545 complex, rather than specific pigment-protein interactions. This is explained by the remarkable conformational flexibility of the eight bilin pigments in PE545, characterized by a quasi-linear arrangement of four pyrrole units. The MD-QM/MM site energies allow us to reproduce the main features of the spectra, and minor adjustments of the energies of the three red-most pigments DBV19A, DBV19B, and PEB82D allow us to model the spectra of PE545 with a similar quality compared to our original model (model E from Novoderezhkin et al. Biophys. J.2010, 99, 344), which was extracted from the spectral and kinetic fit. Moreover, the fit of the transient absorption kinetics is even better in the new structure-based model. The largest difference between our previous and present results is that the MD-QM/MM calculations predict a much smaller gap between the PEB50/61C and PEB50/61D sites, in better accord with chemical intuition. We conclude that the current adjusted MD-QM/MM energies are more reliable in order to explore the spectral properties and energy transfer dynamics in the PE545 complex

Heterogeneity of Signal Transducer and Activator of Transcription Binding Sites in the Long-Terminal Repeats of Distinct HIV-1 Subtypes

HIV-1 can be subdivided into distinct subtypes; the consequences of such a genomic variability remain largely speculative. The long terminal repeats (LTR) control HIV transcription and reflect the major differences of distinct viral subtypes. Three regions in the HIV-1 subtype B LTR are close matches to the Signal Transducer and Activator of Transcription (STAT) consensus sequence. Here, we show heterogeneity in these putative STAT binding sites among HIV-1 LTR subtypes A through G. Transfection of constitutively activated STAT5 lead to transcriptional activation of HIV-1 expression in 293T cells transfected with a reporter assay driven by HIV-1 LTR subtype B. Constitutively activated STAT5 transactivated the LTR of various subtypes in U937 cells with different potency. These findings support and expand the potential relevance of STAT5 activation in HIV infection and may bear relevance for a differential regulation of latency and expression of different subtypes of HIV-1

Cellular origin and microRNA profiles of circulating extracellular vesicles in different stages of diabetic nephropathy

Background: Diabetic nephropathy (DN) is a major complication of diabetes and the main cause of end-stage renal disease. Extracellular vesicles (EVs) are small cell-derived vesicles that can alter disease progression by microRNA (miRNA) transfer. Methods: In this study, we aimed to characterize the cellular origin and miRNA content of EVs in plasma samples of type 2 diabetes patients at various stages of DN. Type 2 diabetes patients were classified in three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. The concentration and cellular origin of plasma EVs were measured by flow cytometry. A total of 752 EV miRNAs were profiled in 18 subjects and differentially expressed miRNAs were validated. Results: Diabetic patients with microalbuminuria and/or macroalbuminuria showed elevated concentrations of total EVs and EVs from endothelial cells, platelets, leucocytes and erythrocytes compared with diabetic controls. miR-99a-5p was upregulated in macroalbuminuric patients compared with normoalbuminuric and microalbuminuric patients. Transfection of miR-99a-5p in cultured human podocytes downregulated mammalian target of rapamycin (mTOR) protein expression and downregulated the podocyte injury marker vimentin. Conclusions: Type 2 diabetes patients with microalbuminuria and macroalbuminuria display differential EV profiles. miR-99a-5p expression is elevated in EVs from macroalbuminuria and mTOR is its validated mRNA target

The native architecture of a photosynthetic membrane

In photosynthesis, the harvesting of solar energy and its subsequent conversion into a stable charge separation are dependent upon an interconnected macromolecular network of membrane-associated chlorophyll–protein complexes. Although the detailed structure of each complex has been determined, the size and organization of this network are unknown. Here we show the use of atomic force microscopy to directly reveal a native bacterial photosynthetic membrane. This first view of any multi-component membrane shows the relative positions and associations of the photosynthetic complexes and reveals crucial new features of the organization of the network: we found that the membrane is divided into specialized domains each with a different network organization and in which one type of complex predominates. Two types of organization were found for the peripheral light-harvesting LH2 complex. In the first, groups of 10–20 molecules of LH2 form light-capture domains that interconnect linear arrays of dimers of core reaction centre (RC)–light-harvesting 1 (RC–LH1–PufX) complexes; in the second they were found outside these arrays in larger clusters. The LH1 complex is ideally positioned to function as an energy collection hub, temporarily storing it before transfer to the RC where photochemistry occurs: the elegant economy of the photosynthetic membrane is demonstrated by the close packing of these linear arrays, which are often only separated by narrow 'energy conduits' of LH2 just two or three complexes wide

We12BFit!-Improving Physical Fitness in 7-12-Year-Old Children With Developmental Coordination Disorder:Protocol of a Multicenter Single-Arm Mixed-Method Study

Background: Children with developmental coordination disorder (DCD) are less physically fit than their typically developing peers. No substantiated treatments are available for children with DCD to address this issue. Aims: This study aims to describe 1. the design and rationale of We12BFit!-PF, a training to increase cardiorespiratory fitness, muscle strength and anaerobic power in 7-12-year-old children with DCD and 2. the methods to examine its preliminary effectiveness and feasibility. Methods: We12BFit!-PF was developed using the steps of defining a treatment theory as proposed by Whyte et al. This includes the definition of targets, mechanisms of action, and essential ingredients. We12BFit!-PF will be evaluated in children diagnosed with DCD according to the criteria of the Diagnostic and Statistical Manual of mental disorders (DSM-V) aged 7-12, recruited from rehabilitation centers and physical therapy clinics. Indication for participation will be a need related to enhancing PF, for example tiring quickly, being quickly out of breath or being unable to keep up with peers during PA. During the treatment the participants will be engaged in a group training (2 × 60 min/week, 10 weeks) targeting cardiorespiratory fitness using high intensity interval training, muscle strength using exercises without weights and anaerobic power using plyometrics. Training intensity during high intensity interval training will be monitored with heart rate monitors, if necessary the intensity will be adjusted. Using a single-arm mixed-method design, the preliminary effectiveness will be determined using the 20 meter Shuttle Run Test, hand held dynamometry (JAMAR and MicroFET) and the Muscle Power Sprint Test, which will be assessed in week 0, 11, and 23. Feasibility will be assessed by interviewing parents and children and by organizing a focus group session with the trainers at the end of We12BFit!-PF. Based on a 5% improvement in VO2peak the minimum sample size is 19 children. Ethics and dissemination: The University of Groningen, University Medical Center Groningen medical ethics committee approved the study (METC 2015.216). Final results will be disseminated via scientific publications, presentations and congress proceedings. Funding organizations will receive a final study report. Trial Registration: This study was registered with Netherlands Trial Registry (NTR6334, www.trialregister.nl)

Spectroscopic characterization of CP26, a chlorophyll a/b binding protein of the higher plant Photosystem II complex

A spectroscopic study is presented of the minor chlorophyll a / b binding protein CP26 isolated from spinach by means of a dodecylmaltoside/betaine washing procedure. The preparations are characterized by a chlorophyll a / chlorophyll b ratio of 3.3 +/- 0.1, and most likely contain 2 chlorophyll b (Chl b) and 6-7 chlorophyll a (Chl a) molecules per monomeric protein. Some of the spectroscopic properties of CP26 show strong similarities to those of the major chlorophyll a / b light-harvesting protein LHC-II, which is in line with the sequence homologies between the two proteins. Spectroscopic differences in the Chl b absorption region are caused by the variation in Chl b content in both proteins. A strongly blue-shifted Chl b band at 637 nm in CP26 has similar linear and circular dichroism properties as the spectral component at 640 nm of LHC-II. It is suggested that these spectral features arise from a conserved Chl b molecule, and that the blue shifts are caused by charged amino acids in the vicinity of these Chl b molecules. The other Chl b band in CP26 is observed at 650 nm. Differences in the Chl a absorption region mainly concern the reduced absorption at 670 nm for CP26, whereas a strong band near 675 nm is very similar to the band at 676 nm for LHC II. Tentative assignments of several absorption bands of CP26 and LHC II to specific pigments are made on the basis of the recently reported three-dimensional structure of LHC II and of the primary amino acid sequences of both proteins. In the carotenoid region LHC II and CP26 show slightly different linear and circular dichroism signals. However, the differences are not large enough to exclude a similar arrangement of the two lutein molecules in LHC II and CP26.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31112/1/0000008.pd