On the front line of HIV virological monitoring: barriers and facilitators from a provider perspective in resource-limited settings.

Scale-up of viral load (VL) monitoring for HIV-infected patients on antiretroviral therapy (ART) is a priority in many resource-limited settings, and ART providers are critical to effective program implementation. We explored provider-perceived barriers and facilitators of VL monitoring. We interviewed all providers (n = 17) engaged in a public health evaluation of dried blood spots for VL monitoring at five ART clinics in Malawi. All ART clinics were housed within district hospitals. We grouped themes at patient, provider, facility, system, and policy levels. Providers emphasized their desire for improved ART monitoring strategies, and frustration in response to restrictive policies for determining which patients were eligible to receive VL monitoring. Although many providers pled for expansion of monitoring to include all persons on ART, regardless of time on ART, the most salient provider-perceived barrier to VL monitoring implementation was the pressure of work associated with monitoring activities. The work burden was exacerbated by inefficient data management systems, highlighting a critical interaction between provider-, facility-, and system-level factors. Lack of integration between laboratory and clinical systems complicated the process for alerting providers when results were available, and these communication gaps were intensified by poor facility connectivity. Centralized second-line ART distribution was also noted as a barrier: providers reported that the time and expenses required for patients to collect second-line ART frequently obstructed referral. However, provider empowerment emerged as an unexpected facilitator of VL monitoring. For many providers, this was the first time they used an objective marker of ART response to guide clinical management. Providers' knowledge of a patient's virological status increased confidence in adherence counseling and clinical decision-making. Results from our study provide unique insight into provider perceptions of VL monitoring and indicate the importance of policies responsive to individual and environmental challenges of VL monitoring program implementation. Findings may inform scale-up by helping policy-makers identify strategies to improve feasibility and sustainability of VL monitoring.

Linker proteins enable ultrafast excitation energy transfer in the phycobilisome antenna system of Thermosynechococcus vulcanus.

We applied a femtosecond flash method, using induced transient absorption changes, to obtain a time-resolved view of excitation energy transfer in intact phycobilisomes of Thermosynechococcus vulcanus at room temperature. Our measurement of an excitation energy transfer rate of 888 fs in phycobilisomes shows the existence of ultrafast kinetics along the phycocyanin rod subcomplex to the allophycocyanin core that is faster than expected for previous excitation energy transfer based on Förster theory in phycobilisomes. Allophycocyanin in the core further transfers energy to the terminal emitter(s) in 17 ps. In the phycobilisome, rod doublets composed of hexameric phycocyanin discs and internal linker proteins are arranged in a parallel fashion, facilitating direct rod-rod interactions. Excitonic splitting likely drives rod absorption at 635 nm as a result of strong coupling between ß84 chromophores (20 ± 1 Å) in adjacent hexamers. In comparison to the absorbance of the phycobilisome antenna system of the cyanobacterium Acaryochloris marina, which possesses a single rod structure, the linkers in T. vulcanus rods induce a 17 nm red shift in the absorbance spectrum. Furthermore, the kinetics of 888 fs indicates that the presence of the linker protein induces ultrafast excitation energy transfer between phycocyanin and allophycocyanin inside the phycobilisome, which is faster than all previous excitation energy transfer in phycobilisome subunits or sub-complexes reported to date.

Induction of apoptosis in human cancer cells by targeting mitochondria with gold nanoparticles.

A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell death. The ability to target Au nanoparticles into mitochondria of breast cancer cells and induce apoptosis reveals an alternative application of Au nanoparticles in photothermal therapy of cancer.

Evidence of additional excitation energy transfer pathways in the phycobiliprotein antenna system of Acaryochloris marina.

To improve the energy conversion efficiency of solar organic cells, the clue may lie in the development of devices inspired by an efficient light harvesting mechanism of some aquatic photosynthetic microorganisms that are adapted to low light intensity. Consequently, we investigated the pathways of excitation energy transfer (EET) from successive light harvesting pigments to the low energy level inside the phycobiliprotein antenna system of Acaryochloris marina, a cyanobacterium, using a time resolved absorption difference spectroscopy with a resolution time of 200 fs. The objective was to understand the actual biochemical process and pathways that determine the EET mechanism. Anisotropy of the EET pathway was calculated from the absorption change trace in order to determine the contribution of excitonic coupling. The results reveal a new electron energy relaxation pathway of 14 ps inside the phycocyanin component, which runs from phycocyanin to the terminal emitter. The bleaching of the 660 nm band suggests a broader absorption of the terminal emitter between 660 nm and 675 nm. Further, there are trimer depolarization kinetics of 450 fs and 500 fs in high and low ionic strength, respectively, which arise from the relaxation of the ß84 and α84 in adjacent monomers of phycocyanin. Under conditions of low ionic strength buffer solution, the evolution of the kinetic amplitude during the depolarization of the trimer is suggestive of trimer conservation within the phycocyanin hexamer. The anisotropy values were 0.38 and 0.40 in high and in low ionic strength, respectively, indicating that there is no excitonic delocalization in the high energy level of phycocyanin hexamers.

Activation and deactivation of vibronic channels in intact phycocyanin rods.

We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm(-1) is assigned to the C-C stretching vibration while the mode at 454 cm(-1) is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm(-1) does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm(-1) rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N-H rocking in-plan vibration.