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Context.--Stanford Pathology began stepwise subspecialty implementation of whole slide imaging (WSI) in 2018 soon after the first US Food and Drug Administration approval. In 2020, during the COVID-19 pandemic, the Centers for Medicare & Medicaid Services waived the requirement for pathologists to perform diagnostic tests in Clinical Laboratory Improvement Amendments (CLIA)-licensed facilities. This encouraged rapid implementation of WSI across all surgical pathology subspecialties. Objective.--To present our experience with validation and implementation of WSI at a large academic medical center encompassing a caseload of more than 50 000 cases per year. Design.--Validation was performed independently for 3 subspecialty services with a diagnostic concordance threshold above 95%. Analysis of user experience, staffing, infrastructure, and information technology was performed after department-wide expansion. Results.--Diagnostic concordance was achieved in 96% of neuropathology cases, 100% of gynecologic pathology cases, and 98% of immunohistochemistry cases. After full implementation, 8 high-capacity scanners were operational, with whole slide images generated on greater than 2000 slides per weekday, accounting for approximately 80% of histologic slides at Stanford Medicine. Multiple modifications in workflow and information technology were needed to improve performance. Within months of full implementation, most attending pathologists and trainees had adopted WSI for primary diagnosis. Conclusions.--WSI across all surgical subspecialities is achievable at scale at an academic medical center; however, adoption required flexibility to adjust workflows and develop tailored solutions. WSI at scale supported the health and safety of medical staff while facilitating high-quality patient care and education during COVID-19 restrictions. doi: 10.5858/arpa.2021-0438-OA, Whole slide imaging (WSI) for digital review of histologic slides in surgical pathology is a revolution in the practice of our craft and has significant operational, diagnostic, and research implications. [...]

* Context.--Pancreatic neuroendocrine tumors (PanNETs) are heterogeneous tumors with a wide range of malignant potential. Therefore, identification of prognostic factors is essential. Objective.--To systematically assess the significance of tumor border, a well-known prognostic indicator in other cancers, in PanNETs. Design.--We evaluated the macroscopic growth pattern (expansile [Exp] versus infiltrative [Inf]) and the microscopic tumor border (pushing [Pus] versus Inf) of 203 surgically resected PanNETs and compared them with other clinicopathologic factors. Results.--Based on macroscopic growth pattern, 83 cases had Exp patterns whereas 84 had Inf patterns. According to microscopic tumor border, 122 PanNETs had Pus borders whereas 81 had Inf borders. Combining macroscopic growth pattern and microscopic tumor border, 65 PanNETs had Exp/Pus, 34 had Inf/Pus, 18 had Exp/Inf, and 50 had Inf/Inf status. PanNETs with Inf/Inf status were associated with higher tumor grade, pT classification, and American Joint Committee on Cancer stage grouping; lymph node metastasis; and lymphovascular and perineural invasions (all P < .001). Patients with PanNET having Inf/Inf status had significantly shorter overall survival (OS) and recurrence-free survival (RFS; all P < .001). Further, using multivariate analysis, Inf/Inf status was identified as an independent poor prognostic factor of OS (P = .02) and RFS (P = .03). Conclusions.--In summary, combined Inf/Inf status was observed in approximately 25% of PanNETs and was associated with aggressive biological behavior and short OS and RFS. Therefore, assessing combined macroscopic growth pattern and microscopic tumor border can provide additional information regarding survival and recurrence in PanNET patients., Pancreatic neuroendocrine tumor (PanNET) is the second most common solid neoplasm of the pancreas, accounting for 3% to 4% of all pancreas neoplasms. (1) PanNET is a heterogeneous tumor composed [...]

Byline: Salecha. Jain, Kinnera. Boina, Suruthi. Purushothaman, Kanmani. Indra Trichoscopy is a non-invasive scalp and hair dermoscopy. In our daily dermatology practice, many eponyms are used. The present article highlights [...]

Byline: Anita. Subudhi, Swapna. Jena, Prasenjeet. Mohanty, Dinesh. Panda Background: As face is the index of the mind, so is the nail the index to health, as the nail is [...]

Byline: Vivek. Tandon, Amol. Raheja, Kanwaljeet. Garg, Shashank. Kale Background: High-speed drilling during neurosurgical procedures lead to aerosol generation. Contaminated particles when dispersed can lead to transmission of disease in [...]

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol [...]

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome. endosome | photooxidation | recycling | readily releasable pool| stimulated emission depletion microscopy doi/ 10.1073/pnas.1007037107

Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy. coherent diffractive imaging | equally sloped tomography | lensless imaging | iterative phase-retrieval algorithms | oversampling doi/ 10.1073/pnas.1000156107

To study how the dynamic subcellular mechanical properties of the heart relate to the fundamental underlying process of actin-myosin cross-bridge cycling, we developed a novel atomic force microscope elastography technique for mapping spatiotemporal stiffness of isolated, spontaneously beating neonatal rat cardiomyocytes. Cells were indented repeatedly at a rate close but unequal to their contractile frequency. The resultant changes in pointwise apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 [+ or -] 5.1 kPa and a diastolic value of 7.8 [+ or -] 4.1 kPa at a representative depth of 400 nm. In cells probed along their major axis, spatiotemporal changes in systolic stiffness displayed a heterogeneous pattern, reflecting the banded sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform apparent modulus of 6.5 [+ or -] 4.8 kPa. This study represents the first quantitative dynamic mechanical mapping of beating cardiomyocytes. The technique provides a means of probing the micromechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes, providing new insights and relating subcellular cardiac structure and function. sarcomere; transverse stiffness; Young's modulus; elasticity; nanoindentation doi:10.1152/ajpheart.00427.2009

The incorporation of viral genomes into particles has never previously been imaged in live infected cells. Thus, for many viruses it is unknown how the recruitment and packaging of genomes into virions is temporally and spatially related to particle assembly. Here, we devised approaches to simultaneously image HIV-1 genomes, as well as the major HIV-1 structural protein, Gag, to reveal their dynamics and functional interactions during the assembly of individual viral particles. In the absence of Gag, HIV-1 RNA was highly dynamic, moving in and out of the proximity of the plasma membrane. Conversely, in the presence of Gag, RNA molecules docked at the membrane where their lateral movement slowed and then ceased as Gag assembled around them and they became irreversibly anchored. Viral genomes were not retained at the membrane when their packaging signals were mutated, nor when expressed with a Gag mutant that was not myristoylated. In the presence of a Gag mutant that retained membrane- and RNA-binding activities but could not assemble into particles, the viral RNA docked at the membrane but continued to drift laterally and then often dissociated from the membrane. These results, which provide visualization of the recruitment and packaging of genomes into individual virus particles, demonstrate that a small number of Gag molecules recruit viral genomes to the plasma membrane where they nucleate the assembly of complete virions. RNA packaging | virion morphogenesis www.pnas.org/cgi/doi/10.1073/pnas.0907364106

Neural crest-specific ablation of BMP type IA receptor (BMPRIA) causes embryonic lethality by embryonic day (E) 12.5, and this was previously postulated to arise from a myocardial defect related to signaling by a small population of cardiac neural crest cells (cNCC) in the epicardium. However, as BMP signaling via cNCC is also required for proper development of the outflow tract cushions, precursors to the semilunar valves, a plausible alternate or additional hypothesis is that heart failure may result from an outflow tract cushion defect. To investigate whether the outflow tract cushions may serve as dynamic valves in regulating hemodynamic function in the early embryo, in this study we used noninvasive ultrasound biomicroscopy-Doppler imaging to quantitatively assess hemodynamic function in mouse embryos with P0-Cre transgene mediated neural crest ablation of Bmpr1a (P0 mutants). Similar to previous studies, the neural crest-deleted Bmpr1a P0 mutants died at ~E12.5, exhibiting persistent truncus arteriosus, thinned myocardium, and congestive heart failure. Surprisingly, our ultrasound analyses showed normal contractile indices, heart rate, and atrioventricular conduction in the P0 mutants. However, reversed diastolic arterial blood flow was detected as early as E11.5, with cardiovascular insufficiency and death rapidly ensuing by E12.5. Quantitative computed tomography showed thinning of the outflow cushions, and this was associated with a marked reduction in cell proliferation. These results suggest BMP signaling to cNCC is required for growth of the outflow tract cushions. This study provides definitive evidence that the outflow cushions perform a valve-like function critical for survival of the early mouse embryo. bone morphogenetic proteins; endocardial cushions; heart defects congenital; heart development; ultrasound biomicroscopy doi: 10.1152/ajpheart.00304.2009.

The expression of the HER-2/neu (HER2) gene, a member of the epidermal growth factor receptor family, has been shown to be a valuable prognostic indicator for breast cancer. However, interobserver variability has been reported in the evaluation of HER2 with immunohistochemistry. It has been suggested that automated computer-based evaluation can provide a consistent and objective evaluation of HER2 expression. In this manuscript, we present an automated method for the quantitative assessment of HER2 using digital microscopy. The method processes microscopy images from tissue slides with a multistage algorithm, including steps of color pixel classification, nuclei segmentation, and cell membrane modeling, and extracts quantitative, continuous measures of cell membrane staining intensity and completeness. A minimum cluster distance classifier merges the features to classify the slides into HER2 categories. An evaluation based on agreement analysis with pathologist-derived HER2 scores, showed good agreement with the provided truth. Agreement varied within the different classes with highest agreement (up to 90%) for positive (3+) slides, and lowest agreement (72%-78%) for equivocal (2+) slides which contained ambiguous scoring. The developed automated method has the potential to be used as a computer aid for the immunohistochemical evaluation of HER2 expression with the objective of increasing observer reproducibility. Index Terms--Biomarker, breast cancer, computer-aided immunohistochemistry, digital microscopy, HER2/neu, pathology.

This new periodic column will help clinicians evaluate their proficiency and gain new knowledge in the general principles of dermoscopy. To assess your diagnostic skills, please assess cases 1 through [...]

During postnatal development, altered sensory experience triggers the rapid reorganization of neuronal responses and connections in sensory neocortex. This experience-dependent plasticity is disrupted by reductions of intracortical inhibition. Little is known about how the responses of inhibitory cells themselves change during plasticity. We investigated the time course of inhibitory cell plasticity in mouse primary visual cortex by using functional two-photon microscopy with single-cell resolution and genetic identification of cell type. Initially, local inhibitory and excitatory cells had similar binocular visual response properties, both favoring the contralateral eye. After 2 days of monocular visual deprivation, excitatory cell responses shifted to favor the open eye, whereas inhibitory cells continued to respond more strongly to the deprived eye. By 4 days of deprivation, inhibitory cell responses shifted to match the faster changes in their excitatory counterparts. These findings reveal a dramatic delay in inhibitory cell plasticity. A minimal linear model reveals that the delay in inhibitory cell plasticity potently accelerates Hebbian plasticity in neighboring excitatory neurons. These findings offer a network-level explanation as to how inhibition regulates the experience-dependent plasticity of neocortex. inhibition | ocular dominance | two-photon microscopy | calcium imaging | cortical plasticity

Hydrogen sulfide ([H.sub.2]S) is an endogenous gaseous mediator with the ability to modulate tissue inflammation and pain. The aim of this study was to determine the effect of an [H.sub.2]S donor ([Na.sub.2]S) on leukocyte-endothelium interactions, blood flow, and pain sensation in acutely inflamed knee joints. Acute arthritis was induced in urethane anesthetized C57bl/6 mice by intra-articular injection of kaolin/carrageenan (24-h recovery), and the effect of local administration of [Na.sub.2]S on leukocyte trafficking was measured by intravital microscopy. Synovial blood flow was measured in inflamed knees by laser Doppler perfusion imaging. Finally, the effect of an intra-articular injection of [Na.sub.2]S on joint pain in control and inflamed rats was determined by hindlimb incapacitance and yon Frey hair algesiometry. Local administration of an [H.sub.2]S donor to inflamed knees caused a dose-dependent reduction in leukocyte adherence and an increase in leukocyte velocity. These effects could be inhibited by coadministration of the ATP-sensitive [K.sup.+] channel blocker glibenclamide. Local administration of [Na.sub.2]S to inflamed joints caused a pronounced vasoconstrictor response: however, there was no observable effect of [Na.sub.2]S on joint pain. These findings establish [H.sub.2]S as a novel signaling molecule in rodent knee joints. [H.sub.2]S exhibits potent anti-inflammatory properties, hut with no detectable effect on joint pain. arthritis; blood flow; intravital microscopy; potassium channels

Using novel interferometric quantitative phase microscopy methods, we demonstrate that the surface integral of the optical phase associated with live cells is invariant to cell water content. Thus, we provide an entirely noninvasive method to measure the nonaqueous content or 'dry mass' of living cells. Given the extremely high stability of the interferometric microscope and the femtogram sensitivity of the method to changes in cellular dry mass, this new technique is not only ideal for quantifying cell growth but also reveals spatially resolved cellular and subcellular dynamics of living cells over many decades in a temporal scale. Specifically, we present quantitative histograms of individual cell mass characterizing the hypertrophic effect of high glucose in a mesangial cell model. In addition, we show that in an epithelial cell model observed for long periods of time, the mean squared displacement data reveal specific information about cellular and subcellular dynamics at various characteristic length and time scales. Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells. phase microscopy; interferometric microscopy; cell growth

Much of the current knowledge of human cardiovascular pathologies and treatment strategies has been gained from understanding the cardiac physioiogies and functions in small animal models, such as mice, rats, and zebrafish. In this paper, we present the development of a high-frame-rate duplex ultrasound biomicroscopy (UBM) capable of B-mode imaging and pulsed-wave (PW) Doppler measurement for in vivo cardiovascular investigation in small animals. A frame rate of 200 frames per second (fps) was accomplished at a view of 5 mm x 8 mm, using a novel high-speed sector probe and specially designed lightweight transducers. In a reduced lateral view of 1.2 mm, a frame rate of 400 fps was achieved to examine more detailed cardiac motion. The UBM utilized transducers with different center frequencies (40-75 MHz) and geometries, which made it useful for various applications in small animal cardiac imaging. The highest spatial resolution the UBM achieved was 25 [micro]m x 56 [micro]m. In addition, the image-guided PW Doppler implemented in the UBM demonstrated the detection of the velocity of a moving wire as low as 0.1 mm/s, and flow in a polyimide tube as small as 200 [micro]m in diameter. Furthermore, the UBM achieved a 15-[micro]V minimal detectable signal and a 60-dB dynamic range using a low-cost PCB-based design. Finally, sample in vivo cardiac images of mouse and zebrafish hearts were given. These results showed that the UBM integrated with B-mode and PW Doppler is useful to investigate the pathophysiological mechanism in the cardiovascular studies.

In phosphorescence quenching microscopy (PQM), the multiple excitation of a reference volume produces the integration of oxygen consumption artifacts caused by individual flashes. We analyzed the performance of two types of PQM instruments to explain reported data on [Po.sub.2] in the microcirculation. The combination of a large excitation area (LEA) and high flash rate produces a large oxygen photoconsumption artifact manifested differently in stationary and flowing fluids. A LEA instrument strongly depresses [Po.sub.2] in a motionless tissue, but less in flowing blood, creating an apparent transmural [Po.sub.2] drop in arterioles. The proposed model explains the mechanisms responsible for producing apparent transmural and longitudinal [Po.sub.2] gradients in arterioles, a [Po.sub.2] rise in venules, a hypothetical high respiration rate in the arteriolar wall and mesenteric tissue, a low [Po.sub.2] in lymphatic microvessels, and both low and uniform tissue [Po.sub.2]. This alternative explanation for reported paradoxical results of [Po.sub.2] distribution in the microcirculation obviates the need to revise the dominant role of capillaries in oxygen transport to tissue. Finding a way to eliminate the photoconsumption artifact is crucial for accurate microscopic oxygen measurements in microvascular networks and tissue. The PQM technique that employs a small excitation area (SEA) together with a low flash rate was specially designed to avoid accumulated oxygen photoconsumption in flowing blood and lymph. The related scanning SEA instrument provides artifact-free [Po.sub.2] measurements in stationary tissue and motionless fluids. Thus the SEA technique significantly improves the accuracy of microscopic [Po.sub.2] measurements in the microcirculation using the PQM. oxygen consumption; oxygen tension; oxygen gradient

Reduced sinusoidal endothelial nitric oxide (NO) production contributes to increased intrahepatic resistance and portal hypertension after liver injury. We hypothesized that V-PYRRO/NO, an NO donor prodrug metabolized 'specifically' in the liver, would reduce portal venous pressure (PVP) without affecting the systemic vasculature. Liver injury was induced in male BALB/c mice by weekly C[Cl.sub.4] gavage. PVP and mean arterial pressure were recorded during intravenous administration of V-PYRRO/NO. In vivo microscopy was used to monitor sinusoidal diameter and flow during drug administration. Mean PVP was increased in C[Cl.sub.4]-treated mice compared with sham-treated mice. In dose-response experiments, the minimum dose of PYRRO/NO required to acutely lower PVP by 20%, the amount believed to yield a clinically meaningful outcome, was 200 nmol/kg. This dose decreased portal pressure in cirrhotic (23.4 [+ or -] 2.0%, P < 0.001 vs. vehicle) and sham-treated (19.5 [+ or -] 2.3%, P < 0.001 vs. vehicle) animals by a similar magnitude. This concentration also led to dilation of hepatic sinusoids and an increase in sinusoidal volumetric flow, consistent with a reduction of intrahepatic resistance. The effect of V-PYRRO/NO on mean arterial pressure was significant at all concentrations tested, including the lowest, 30 nmol/kg (P < 0.001 vs. vehicle for all doses). We conclude that V-PYRRO/NO had widespread vascular effects and, as such, is unlikely to be suitable for treatment of portal hypertension. As the potential of this or other similar compounds for treatment of portal hypertension is evaluated, effects on the systemic vasculature will also need to be considered. liver; portal hypertension; sinusoidal resistance; sinusoidal flow; vasculature; in vivo microscopy

Time-lapse microscopy has advanced our understanding of yolk sac and early embryonic vascularization. However, it has been difficult to assess endothelial interactions during epithelial morphogenesis of internal organs. To address this issue we have developed the first time-lapse system to study vascularization of a mammalian organ in four dimensions. We show that vascularization of XX and XY gonads is a highly dynamic, sexually dimorphic process. The XX gonad recruits vasculature by a typical angiogenic process. In contrast, the XY gonad recruits and patterns vasculature by a novel remodeling mechanism beginning with breakdown of an existing mesonephric vessel. Subsequently, in XY organs individual endothelial cells migrate and reaggregate in the coelomic domain to form the major testicular artery. Migrating endothelial cells respect domain boundaries well before they are morphologically evident, subdividing the gonad into 10 avascular regions where testis cords form. This model of vascular development in an internal organ has a direct impact on the current dogma of vascular integration during organ development and presents important parallels with mechanisms of tumor vascularization. organogenesis | ovary | testis

Colocalization, in which images of two or more fluorescent markers are overlaid, and coincidence between the probes is measured or displayed, is a common analytical tool in cell biology. Interpreting the images and the meaning of this identified coincidence is difficult in the absence of basic information about the acquisition parameters. In this commentary, we highlight important factors in the acquisition of images used to demonstrate colocalization, and we discuss the minimum information that authors should include in a manuscript so that a reader can interpret both the fluorescent images and any observed colocalization.

Objective To evaluate the relative cost-effectiveness in different sub-Saharan African settings of presumptive treatment, field-standard microscopy and rapid diagnostic tests (RDTs) to diagnose malaria. Methods We used a decision tree model and probabilistic sensitivity analysis applied to outpatients presenting at rural health facilities with suspected malaria. Costs and effects encompassed those for both patients positive on RDT (assuming artemisinin-based combination therapy) and febrile patients negative on RDT (assuming antibiotic treatment). Interventions were defined as cost-effective if they were less costly and more effective or had an incremental cost per disability-adjusted life year averted of less than US$ 150. Data were drawn from published and unpublished sources, supplemented with expert opinion. Findings RDTs were cost-effective compared with presumptive treatment up to high prevalences of Plasmodium falciparum parasitaemia. Decision-makers can be at least 50% confident of this result below 81% malaria prevalence, and 95% confident below 62% prevalence, a level seldom exceeded in practice. RDTs were more than 50% likely to be cost-saving below 58% prevalence. Relative to microscopy, RDTs were more than 85% likely to be cost-effective across all prevalence levels, reflecting their expected better accuracy under real-life conditions. Results were robust to extensive sensitivity analysis. The cost-effectiveness of RDTs mainly reflected improved treatment and health outcomes for non-malarial febrile illness, plus savings in antimalarial drug costs. Results were dependent on the assumption that prescribers used test results to guide treatment decisions. Conclusion RDTs have the potential to be cost-effective in most parts of sub-Saharan Africa. Appropriate management of malaria and non-malarial febrile illnesses is required to reap the full benefits of these tests. Bulletin of the World Health Organization 2008;86:101-110. Objectif Evaluer le rapport cout-efficacite dans differents pays d&apos;Afrique subsaharienne du traitement presomptif, de la microscopie classique sur le terrain et des tests diagnostiques rapides (TDR) dans le diagnostic du paludisme. Methodes Nous avons fait appel a un module d&apos;arbre de decisions et a une analyse probabiliste de sensibilite, qui ont ete appliques aux patients se presentant en ambulatoire dans des 6tablissements de soins ruraux avec une presomption de paludisme. Nous avons evalue les couts et les effets du traitement la fois pour les patients positifs aux TDR (en les supposant sous traitement par une polytherapie a base d&apos;artemisinine) et les patients febriles negatifs a ces tests (en les supposant sous traitement antibiotique). Nous avons consider que les interventions etalent efficientes sous rangle economique si elles etaient moins couteuses et plus efficaces ou si elles fournissaient un coot marginal par annee de vie corrigee de I&apos;incapacite evite inferieur US $150. Les donnees ont ete extraites de sources publiees et non publiees, completees par des avis d&apos;experts. Resultats Les TDR se sont reveles efficaces sur le plan economique par comparaison avec le traitement presomptif jusqu&apos;a des valeurs elevees de la parasitemie due a Plasmodium falciparum. Les d6cideurs peuvent accorder a ce resultat un niveau de confiance de 50 % lorsque la prevalence du paludisme est inferieure a 81% et de 95 % lorsque cette prevalence est infeieure a 62 %, niveau rarement depasse dans la pratique. Au-dessous de 58 % de prevalence, la probabilite que les TDR permettent des economies etait superieure a 50 %. Par rapport la microscopie, les tests avaient une probabilite de plus de 85 % d&apos;avoir un bon ratio cout-efficacite sur I&apos;ensemble des niveaux de prevalence, en accord avec la meilleure precision qu&apos;on s&apos;attend a leur voir fournir en conditions reelles. Ces resultats ont resiste a une analyse de sensibilite approfondie. Le rapport cout-efficacite des TDR refletait principalement I&apos;amelioration des traitements et des issues des maladies febriles non palustres, ainsi que les economies realisees sur les coots des antipaludiques. Ces resultats supposaient que les prescripteurs aient utilise les reponses des tests pour guider les decisions therapeutiques. Conclusion Les TDR sont en mesure d&apos;avoir un bon rapport coutefficacite dans la plupart des regions de l&apos;Afrique subsaharienne. Une prise en charge appropriee du paludisme et des maladies febriles non palustres est necessaire pour tirer un benefice maximal de ces tests. Objetivo Evaluar en diferentes entornos subsaharianos la costoeficacia relativa del tratamiento de sospecha, la microscopia estandar de campo y las pruebas de diagnostico rapido (PDR) de la malaria. Metodos Se aplico un modelo de arbol decisional y un analisis de sensibilidad probabilistico a pacientes ambulatorios que acudieron a establecimientos de salud rurales con presunta malaria. Se consideraron los costos y los efectos tanto para los pacientes positivos en la PDR (suponiendo que se administraron tratamiento combinado con artemisinina) como para los pacientes febriles negativos en la PDR (suponiendo que se administraron antibioticos). Se definieron como costoeficaces las intervenciones que fueron menos costosas y mas eficaces o que habfan tenido un costo marginal por AVAD (ano de vida ajustado en funcion de la discapacidad) ganado de menos de US$150. Los datos empleados se extrajeron de trabajos publicados o ineditos y se complementaron con la opinion de expertos. Resultados Las PDR fueron costoeficaces en comparacion con el tratamiento de sospecha hasta valores altos de la prevalencia de parasitemia por Plasmodium falciparum. Las instancias decisorias pueden confiar con una probabilidad de al menos un 50% en ese resultado por debajo de una prevalencia de malaria del 81%, y del 95% por debajo de una prevalencia del 62%, nivel rara vez superado en la practica. Las PDR tenian una probabilidad mayor del 50% de ser costoeficaces por debajo de una prevalencia del 58%. En lo que respecta a la microscopia, las PDR tenian mas de un 85% de probabilidades de ser costoeficaces en todos los niveles de prevalencia, lo que refleja la mayor exactitud previsible en condiciones reales. Un extenso analisis de sensibilidad revelo que los resultados eran robustos. La costoeficacia de las PDR se debio principalmente al mejor tratamiento y resultados sanitarios conseguidos con las dolencias febriles no malaricas, mas lo ahorrado en concepto de medicamentos antimalaricos. Los resultados dependen del supuesto de que los prescriptores tomaron sus decisiones terapeuticas en funcion de los resultados de las pruebas. Conclusion Las PDR pueden ser costoeficaces en la mayoria del Africa subsahariana, pero para poder sacar el maximo provecho de esas pruebas hay que tratar adecuadamente la malaria y las enfermedades febriles no malaricas., Introduction The introduction of high-cost antimalarial drugs such as artemisinin-based combination therapy (ACT) is encouraging malaria-endemic countries in sub-Saharan Africa to reassess diagnostic practices. Traditional practice for outpatients has been [...]

Fluorescence correlation spectroscopy (FCS), fluorescence crosscorrelation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with [K.sub.d] >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation. fluorescence correlation spectroscopy p fluorescence cross-correlation spectroscopy I photon-counting histograms I scaffold I fluorescence resonance energy transfer

T cells interacting with antigen-presenting cells (APCs) form an 'immunological synapse' (IS), a bull's-eye pattern composed of a central supramolecular activation cluster enriched with T cell receptors (TCRs) surrounded by a ring of adhesion molecules (a peripheral supramolecular activation cluster). The mechanism responsible for segregating TCR and adhesion molecules remains poorly understood. Here, we show that immortalized Jurkat T cells interacting with a planar lipid bilayer (mimicking an APC) will form an IS, thereby providing an accessible model system for studying the cell biological processes underlying IS formation. We found that an actin-dependent process caused TCR and adhesion proteins to cluster at the cell periphery, but these molecules appeared to segregate from one another at the earliest stages of microdomain formation. The TCR and adhesion microdomains attached to actin and were carried centripetally by retrograde flow. However, only the TCR microdomains penetrated into the actin-depleted cell center, whereas the adhesion microdomains appeared to be unstable without an underlying actin cytoskeleton. Our results reveal that TCR and adhesion molecules spatially partition from one another well before the formation of a mature IS and that differential actin interactions help to shape and maintain the final bull's-eye pattern of the IS. actin | membrane microdomain | planar lipid bilayer | single-molecule imaging | supramolecular activation cluster