Is the Differentiated Service Delivery Model Suited to the Needs of People Living with HIV in Rwanda?

The primary goal of antiretroviral treatment is to improve the health of individuals with HIV, and a secondary goal is to prevent further transmission. In 2016, Rwanda adopted the World Health Organization's "treat-all" approach in combination with the differentiated service delivery (DSD) model. The model's goal was to shorten the time from HIV diagnosis to treatment initiation, regardless of the CD4 T-cell count. This study sought to identify perceptions, enablers, and challenges associated with DSD model adoption among PLHIV.This study included selected health centers in Kigali city, Rwanda, between August and September 2022. The patients included were those exposed to the new HIV care model (DSD) model and those exposed to the previous model who transitioned to the current model. Interviews and focus group discussions were also held to obtain views and opinions on the DSD model. The data were collected via questionnaires and audio-recorded focus group discussions and were subsequently analyzed.The study identified several themes, including participants' initial emotions about a new HIV diagnosis, disclosure, experiences with transitioning to the DSD model, the effect of peer education, and barriers to and facilitators of the DSD model. Participants appreciated reduced clinic visits under the DSD model but faced transition and peer educator mobility challenges.The DSD model reduces waiting times, educates patients, and aligns with national goals. Identified barriers call for training and improved peer educator retention. Recommendations include enhancing the DSD model and future research to evaluate its long-term impact and cost-effectiveness.

Cryofixation during live-imaging enables millisecond time-correlated light and electron microscopy.

Correlating live-cell imaging with electron microscopy is among the most promising approaches to relate dynamic functions of cells or small organisms to their underlying ultrastructure. The time correlation between light and electron micrographs, however, is limited by the sample handling and fixation required for electron microscopy. Current cryofixation methods require a sample transfer step from the light microscope to a dedicated instrument for cryofixation. This transfer step introduces a time lapse of one second or more between live imaging and the fixed state, which is studied by electron microscopy. In this work, we cryofix Caenorhabditis elegans directly within the light microscope field of view, enabling millisecond time-correlated live imaging and electron microscopy. With our approach, the time-correlation is limited only by the sample cooling rate. C. elegans was used as a model system to establish compatibility of in situ cryofixation and subsequent transmission electron microscopy (TEM). TEM images of in situ cryofixed C. elegans show that the ultrastructure of the sample was well preserved with this method. We expect that the ability to correlate live imaging and electron microscopy at the millisecond scale will enable new paradigms to study biological processes across length scales based on real-time selection and arrest of a desired state. LAY DESCRIPTION: Researchers seek to link cellular functions to their smallest structural components. Currently this requires correlation of two imaging techniques, live imaging and electron microscopy. Current correlative methods, however, have limited time resolution due to the sample preparation procedures for electron microscopy. Following live imaging, samples are transferred from the light microscope to a cryofixation, or ultra-fast freezing, instrument. The biological process progresses until the sample freezes, 1 second or more after the last live image. In this work, samples are cryofixed directly within the light microscope field of view. By eliminating the transfer step, time correlation between light and electron microscopy images of our samples is limited only by the freezing rate to the order of milliseconds rather than seconds.

Resonant addressing and manipulation of silicon vacancy qubits in silicon carbide.

Several systems in the solid state have been suggested as promising candidates for spin-based quantum information processing. In spite of significant progress during the last decade, there is a search for new systems with higher potential [D. DiVincenzo, Nat. Mater. 9, 468 (2010)]. We report that silicon vacancy defects in silicon carbide comprise the technological advantages of semiconductor quantum dots and the unique spin properties of the nitrogen-vacancy defects in diamond. Similar to atoms, the silicon vacancy qubits can be controlled under the double radio-optical resonance conditions, allowing for their selective addressing and manipulation. Furthermore, we reveal their long spin memory using pulsed magnetic resonance technique. All these results make silicon vacancy defects in silicon carbide very attractive for quantum applications.

Synaptic vesicles studied by dynamic light scattering.

The size polydispersity distribution of synaptic vesicles (SVs) is characterized under quasi-physiological conditions by dynamic light scattering (DLS). Highly purified fractions of SVs obtained from rat brain still contain a small amount of larger contaminant structures, which can be quantified by DLS and further reduced by asymmetric-flow field-flow (AFFF) fractionation. The intensity autocorrelation functions g (2)(τ) recorded from these samples are analyzed by a constrained regularization method as well as by an alternative direct modeling approach. The results are in quantitative agreement with the polydispersity obtained from cryogenic electron microscopy of vitrified SVs. Next, different vesicle fusion assays based on samples composed of SVs and small unilamellar proteoliposomes with the fusion proteins syntaxin 1 and SNAP-25A are characterized by DLS. The size increase of the proteoliposomes due to SNARE-dependent fusion with SVs is quantified by DLS under quasi-physiological conditions.

Nonlocal activation of a bistable atom through a surface state charge-transfer process on Si(100)-(2×1):H.

The reversible hopping of a bistable atom on the Si(100)-(2×1):H surface is activated nonlocally by hole injection into Si-Si bond surface states with a low temperature (5 K) scanning tunneling microscope. In the contact region, at short distances (<1.5 nm) between the hole injection site and the bistable atom, the hopping yield of the bistable atom exhibits remarkable variations as a function of the hole injection site. It is explained by the density of state distribution along the silicon bond network that shows charge-transfer pathways between the injection sites and the bistable atom.

NIMROD: The Near and InterMediate Range Order Diffractometer of the ISIS second target station.

NIMROD is the Near and InterMediate Range Order Diffractometer of the ISIS second target station. Its design is optimized for structural studies of disordered materials and liquids on a continuous length scale that extends from the atomic, upward of 30 nm, while maintaining subatomic distance resolution. This capability is achieved by matching a low and wider angle array of high efficiency neutron scintillation detectors to the broad band-pass radiation delivered by a hybrid liquid water and liquid hydrogen neutron moderator assembly. The capabilities of the instrument bridge the gap between conventional small angle neutron scattering and wide angle diffraction through the use of a common calibration procedure for the entire length scale. This allows the instrument to obtain information on nanoscale systems and processes that are quantitatively linked to the local atomic and molecular order of the materials under investigation.

Fluorescence nanoscopy with optical sectioning by two-photon induced molecular switching using continuous-wave lasers.

During the last decade far-field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous-wave laser beam. This bright marker enables strict control of on/off switching and provides single-molecule localization precision down to 15 nm in the focal plane. Two-photon induced nonlinear photoswitching of this marker with continuous-wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost-effective fluorescence nanoscopy with noninvasive optical sectioning.

Rhodotorula glutinis fungemia in a liver-kidney transplant patient.

A 54-year-old man underwent simultaneous liver-kidney transplantation. During his prolonged hospitalization, he developed catheter-related fungemia with Rhodotorula glutinis and azole-resistant Candida glabrata. Management of the Rhodotorula fungemia was complicated by his renal insufficiency, hepatic insufficiency, and the concurrent fungemia with multi-azole resistant C. glabrata. He was treated with combination therapy with voriconazole and micafungin with subsequent clearance of the fungemia. Rhodotorula species are emerging as human pathogens with the increasing number of immunosuppressed patients in the last few decades. This is the first report of a R. glutinis fungemia in a solid organ transplant recipient.

Mastering the molecular dynamics of a bistable molecule by single atom manipulation.

At low temperature (5 K), a single biphenyl molecule adsorbed on a Si(100) surface behaves as a bistable device which can be reversibly switched by electronic excitation with the scanning tunneling microscope tip. Density functional theory suggests that the biphenyl molecule is adsorbed with one dissociated hydrogen atom bonded to a neighbor surface silicon atom. By desorbing this hydrogen atom with the STM tip, the interaction of the molecule with the surface is modified such that it becomes transformed into a multistable device with four stable states having switching yields increased by almost 2 orders of magnitude.

Screening for human immunodeficiency virus (HIV) dementia in an HIV clade C-infected population in India.

Although human immunodeficiency virus (HIV) clade C virus infects the largest populations worldwide, to date there are no prospective studies reported thus far to determine the incidence or prevalence of HIV dementia in this population. HIV clade C virus is a CCR5-tropic virus and thus predominantly infects macrophages, which are the key cells implicated in the pathogenesis of HIV dementia. However, HIV dementia has only rarely been reported in these populations. The authors thus used a recently developed International HIV Dementia Scale (IHDS) to screen a well-characterized cohort of HIV-infected discordant couples in Pune, India. 48 HIV+ subjects with CD4 cell count <200 cells/mm(3) and 48 HIV- subjects were studied. The HIV+ subjects had significantly lower IHDS scores compared to the HIV- subjects. 35% of the HIV+ subjects and 15% of the HIV- subjects scored < 10 on the IHDS. These observations suggest that the prevalence of HIV dementia may be higher in this population than previously reported. More importantly, it demonstrates that the IHDS can be used as a screening tool in the Indian population.

Picometer-scale electronic control of molecular dynamics inside a single molecule.

Tunneling electrons from a low-temperature (5 kelvin) scanning tunneling microscope were used to control, through resonant electronic excitation, the molecular dynamics of an individual biphenyl molecule adsorbed on a silicon(100) surface. Different reversible molecular movements were selectively activated by tuning the electron energy and by selecting precise locations for the excitation inside the molecule. Both the spatial selectivity and energy dependence of the electronic control are supported by spectroscopic measurements with the scanning tunneling microscope. These experiments demonstrate the feasibility of controlling the molecular dynamics of a single molecule through the localization of the electronic excitation inside the molecule.

Atomic-scale STM experiments on semiconductor surfaces: towards molecular nanomachines.

The electronic or quantum control of individual molecules with the scanning tunnelling microscope offers exciting perspectives on operating molecular nanomachines. This implies the use of semiconductor surfaces rather than metallic surfaces which would rapidly quench the electronic excitations. We review recent results illustrating the state of the art and the main problems which need to be solved: the choice, design and properties of functionalized organic molecules on semiconductor surfaces; the control of the inelastic electronic channels through a single molecule; and the search for well-controlled atomic-scale wide-band-gap semiconductor surfaces.

SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis.

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.

Exocytosis requires asymmetry in the central layer of the SNARE complex.

Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Quantal release of serotonin.

We have studied the origin of quantal variability for small synaptic vesicles (SSVs) and large dense-cored vesicles (LDCVs). As a model, we used serotonergic Retzius neurons of leech that allow for combined amperometrical and morphological analyses of quantal transmitter release. We find that the transmitter amount released by a SSV varies proportionally to the volume of the vesicle, suggesting that serotonin is stored at a constant intravesicular concentration and is completely discharged during exocytosis. Transmitter discharge from LDCVs shows a higher degree of variability than is expected from their size distribution, and bulk release from LDCVs is slower than release from SSVs. On average, differences in the transmitter amount released from SSVs and LDCVs are proportional to the size differences of the organelles, suggesting that transmitter is stored at similar concentrations in SSVs and LDCVs.

The SNARE Vti1a-beta is localized to small synaptic vesicles and participates in a novel SNARE complex.

Specific soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins are required for different membrane transport steps. The SNARE Vti1a has been colocalized with Golgi markers and Vti1b with Golgi and the trans-Golgi network or endosomal markers in fibroblast cell lines. Here we study the distribution of Vti1a and Vti1b in brain. Vti1b was found in synaptic vesicles but was not enriched in this organelle. A brain-specific splice variant of Vti1a was identified that had an insertion of seven amino acid residues next to the putative SNARE-interacting helix. This Vti1a-beta was enriched in small synaptic vesicles and clathrin-coated vesicles isolated from nerve terminals. Vti1a-beta also copurified with the synaptic vesicle R-SNARE synaptobrevin during immunoisolation of synaptic vesicles and endosomes. Therefore, both synaptobrevin and Vti1a-beta are integral parts of synaptic vesicles throughout their life cycle. Vti1a-beta was part of a SNARE complex in nerve terminals, which bound N-ethylmaleimide-sensitive factor and alpha-SNAP. This SNARE complex was different from the exocytic SNARE complex because Vti1a-beta was not coimmunoprecipitated with syntaxin 1 or SNAP-25. These data suggest that Vti1a-beta does not function in exocytosis but in a separate SNARE complex in a membrane fusion step during recycling or biogenesis of synaptic vesicles.

Differential tolerance induction by lipoarabinomannan and lipopolysaccharide in human macrophages.

Various bacterial cell wall components have been shown to induce hyporesponsiveness in macrophages (MAC). Here, mycobacterial glycolipids were employed to determine whether they induce a state of 'tolerance/hyporesponsiveness' in MAC in vitro in order to assess whether mycobacterial components negatively affect the immune response to Mycobacterium tuberculosis. Arabinosylated lipoarabinomannan (ARA-LAM) stimulated hyporesponsiveness by reducing TNF-alpha, GM-CSF, G-CSF, IL-10, and IL-6 release similarly to LPS, but caused no changes in IL-8 secretion. Mannose-capped LAM (MAN-LAM) acted in a different way in that TNF-alpha, GM-CSF, and IL-10 were upregulated after restimulation of MAC. Blocking experiments by mannan suggest mannose-receptor involvement in MAN-LAM activation only. Cross-stimulation experiments demonstrated a hierarchy of signaling, with LPS being the most potent stimulator and mediating abrogation of ARA-LAM-stimulated tolerance but not vice versa. MAN-LAM was the least potent stimulator of either MAC activation and induction of hyporesponsiveness. Similarly to LPS, ARA-LAM upregulated CD14 surface expression after restimulation. Recurrent MAN-LAM treatment either downmodulated or did not induce any change in CD14 expression. The role of MAN-LAM regulated cytokine secretion as well as implications regarding M. tuberculosis infection will be discussed.

Immunoisolation of GABA-specific synaptic vesicles defines a functionally distinct subset of synaptic vesicles.

Synaptic vesicles from mammalian brain are among the best characterized trafficking organelles. However, so far it has not been possible to characterize vesicle subpopulations that are specific for a given neurotransmitter. Taking advantage of the recent molecular characterization of vesicular neurotransmitter transporters, we have used an antibody specific for the vesicular GABA transporter (VGAT) to isolate GABA-specific synaptic vesicles. The isolated vesicles are of exceptional purity as judged by electron microscopy. Immunoblotting revealed that isolated vesicles contain most of the major synaptic vesicle proteins in addition to VGAT and are devoid of vesicular monoamine and acetylcholine transporters. The vesicles are 10-fold enriched in GABA uptake activity when compared with the starting vesicle fraction. Furthermore, glutamate uptake activity and glutamate-induced but not chloride-induced acidification are selectively lost during immunoisolation. We conclude that the population of GABA-containing synaptic vesicles is separable and distinct from vesicle populations transporting other neurotransmitters.

A cell-free system for regulated exocytosis in PC12 cells.

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.