Model-based wavefront shaping microscopy

Wavefront shaping is increasingly being used in modern microscopy to obtain distortion-free, high-resolution images deep inside inhomogeneous media. Wavefront shaping methods typically rely on the presence of a 'guidestar' in order to find the optimal wavefront to mitigate the scattering of light. However, this condition cannot be satisfied in most biomedical applications. Here, we introduce a novel, guidestar-free wavefront shaping method in which the optimal wavefront is computed using a digital model of the sample. The refractive index model of the sample, that serves as the input for the computation, is constructed in-situ by the microscope itself. In a proof of principle imaging experiment, we demonstrate a large improvement in the two-photon fluorescence signal through a diffuse medium, outperforming the state-of-the-art wavefront shaping techniques by a factor of 21

Perfusion measured by laser speckle contrast imaging as a predictor for expansion of psoriasis lesions

BACKGROUND: Skin microvasculature changes are crucial in psoriasis development and correlate with perfusion. The noninvasive Handheld Perfusion Imager (HAPI) examines microvascular skin perfusion in large body areas using laser speckle contrast imaging (LSCI). OBJECTIVES: To (i) assess whether increased perilesional perfusion and perfusion inhomogeneity are predictors for expansion of psoriasis lesions and (ii) assess feasibility of the HAPI system in a mounted modality. METHODS: In this interventional pilot study in adults with unstable plaque psoriasis, HAPI measurements and color photographs were performed for lesions present on one body region at week 0, 2, 4, 6 and 8. The presence of increased perilesional perfusion and perfusion inhomogeneity was determined. Clinical outcome was categorized as increased, stable or decreased lesion surface between visits. Patient feedback was collected on a 10‐point scale. RESULTS: In total, 110 lesions with a median follow‐up of 6 (IQR 6.0) weeks were assessed in 6 patients with unstable plaque psoriasis. Perfusion data was matched to 281 clinical outcomes after two weeks. A mixed multinomial logistic regression model revealed a predictive value of perilesional increased perfusion (OR 9.90; p < 0.001) and perfusion inhomogeneity (OR 2.39; p = 0.027) on lesion expansion after two weeks compared to lesion stability. HAPI measurements were considered fast, patient‐friendly and important by patients. CONCLUSION: Visualization of increased perilesional perfusion and perfusion inhomogeneity by noninvasive whole field LSCI holds potential for prediction of psoriatic lesion expansion. Furthermore, the HAPI is a feasible and patient‐friendly tool

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.

Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis

STXBP1 promotes Weibel-Palade body exocytosis through its interaction with the Rab27A effector Slp4-a.

Vascular endothelial cells contain unique rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs), which contain the hemostatic protein von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormone-evoked WPB exocytosis. Using a nonbiased proteomic screen for targets for Slp4-a, we now identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Coimmunoprecipitations showed that STXBP1 interacts with syntaxin-2 and -3, but not with syntaxin-4. Small interfering RNA-mediated silencing of STXBP1 expression impaired histamine- and forskolin-induced VWF secretion. To further substantiate the role of STXBP1, we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1-haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared with control BOECs of healthy donors; however, EIEE4 BOECs displayed significantly impaired histamine- and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature

The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites