Using a programmatic mapping approach to plan for HIV prevention and harm reduction interventions for people who inject drugs in three South African cities.

BACKGROUND: Stigma, criminalisation and a lack of data on drug use contribute to the "invisibility" of people who inject drugs (PWID) and make HIV prevention and treatment service delivery challenging. We aimed to confirm locations where PWID congregate in Cape Town, eThekwini and Tshwane (South Africa) and to estimate PWID population sizes within selected electoral wards in these areas to inform South Africa's first multi-site HIV prevention project for PWID. METHODS: Field workers (including PWID peers) interviewed community informants to identify suspected injecting locations in selected electoral wards in each city and then visited these locations and interviewed PWID. Interviews were used to gather information about the accessibility of sterile injecting equipment, location coordinates and movement patterns. We used the Delphi method to obtain final population size estimates for the mapped wards based on estimates from wisdom of the crowd methods, the literature and programmatic data. RESULTS: Between January and April 2015, we mapped 45 wards. Tshwane teams interviewed 39 PWID in 12 wards, resulting in an estimated number of accessible PWID ranging from 568 to 1431. In eThekwini, teams interviewed 40 PWID in 15 wards with an estimated number of accessible PWID ranging from 184 to 350. The Cape Town team interviewed 61 PWID in 18 wards with an estimated number of accessible PWID ranging between 398 and 503. Sterile needles were only available at one location. Almost all needles were bought from pharmacies. Between 80 and 86% of PWID frequented more than one location per day. PWID who reported movement visited a median of three locations a day. CONCLUSIONS: Programmatic mapping led by PWID peers can be used effectively to identify and reach PWID and build relationships where access to HIV prevention commodities for PWID is limited. PWID reported limited access to sterile injecting equipment, highlighting an important HIV prevention need. Programmatic mapping data show that outreach programmes should be flexible and account for the mobile nature of PWID populations. The PWID population size estimates can be used to develop service delivery targets and as baseline measures.

Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones.

Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase-potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.

Myosin II activity facilitates microtubule bundling in the neuronal growth cone neck.

The cell biological processes underlying axon growth and guidance are still not well understood. An outstanding question is how a new segment of the axon shaft is formed in the wake of neuronal growth cone advance. For this to occur, the highly dynamic, splayed-out microtubule (MT) arrays characteristic of the growth cone must be consolidated (bundled together) to form the core of the axon shaft. MT-associated proteins stabilize bundled MTs, but how individual MTs are brought together for initial bundling is unknown. Here, we show that laterally moving actin arcs, which are myosin II-driven contractile structures, interact with growing MTs and transport them from the sides of the growth cone into the central domain. Upon Myosin II inhibition, the movement of actin filaments and MTs immediately stopped and MTs unbundled. Thus, Myosin II-dependent compressive force is necessary for normal MT bundling in the growth cone neck.

Myosin II functions in actin-bundle turnover in neuronal growth cones.

Retrograde actin flow works in concert with cell adhesion to generate traction forces that are involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but identification of the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report that inhibition of myosin II alone decreases retrograde flow by 51% and the remaining flow can be almost fully accounted for by the 'push' of plus-end actin assembly at the leading edge of the growth cone. Interestingly, actin bundles that are associated with filopodium roots elongated by approximately 83% after inhibition of myosin II. This unexpected result was due to decreased rates of actin-bundle severing near their proximal (minus or pointed) ends which are located in the transition zone of the growth cone. Our study reveals a mechanism for the regulation of actin-bundle length by myosin II that is dependent on actin-bundle severing, and demonstrate that retrograde flow is a steady state that depends on both myosin II contractility and actin-network treadmilling.