Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) assay is the current model for assessment of human normal and leukemic stem cells. We explored why 51% of 59 acute myeloid leukemia (AML) patients were unable to initiate leukemia in NOD/SCID mice. Increasing the cell dose, using more permissive recipients, and alternative tissue sources did not cause AML engraftment in most previously nonengrafting AML samples. Homing of AML cells to the marrow was the same between engrafters and nonengrafters. FLT3 internal tandem duplication (ITD) and nucleophosmin mutations occurred at a similar frequency in engrafters and nonengrafters. The only variable that was related to engraftment ability was the karyotypically defined risk stratification of individual AML cases. Of interest, follow-up of younger patients with intermediate-risk AML revealed a significant difference in overall survival between NOD/SCID engrafting and nonengrafting AMLs. Hence, the ability of AML to engraft in the NOD/SCID assay seems to be an inherent property of AML cells, independent of homing, conditioning, or cell frequency/source, which is directly related to prognosis. Our results suggest an important difference between leukemic initiating cells between engrafting and nonengrafting AML cases that correlates with treatment response.

In its classical view, the renin angiotensin system (RAS) was defined as an endocrine system involved in blood pressure regulation and body electrolyte balance. However, the emerging concept of tissue RAS, along with the discovery of new RAS components, increased the physiological and clinical relevance of the system. Indeed, RAS has been shown to be expressed in various tissues where alterations in its expression were shown to be involved in multiple diseases including atherosclerosis, cardiac hypertrophy, type 2 diabetes (T2D) and renal fibrosis. In this chapter, we describe the new components of RAS, their tissue-specific expression, and their alterations under pathological conditions, which will help achieve more tissue- and condition-specific treatments.

A rapid and reliable diagnostic test to distinguish ischemic from hemorrhagic stroke in patients presenting with stroke-like symptoms is essential to optimize management and triage for thrombolytic therapy. The present study measured serum concentrations of ubiquitin C-terminal hydrolase (UCH-L1) and glial fibrillary astrocytic protein (GFAP) in acute stroke patients and healthy controls and investigated their relation to stroke severity and patient characteristics. We also assessed the diagnostic performance of these markers for the differentiation of intracerebral hemorrhage (ICH) from ischemic stroke (IS). Both UCH-L1 and GFAP concentrations were significantly greater in ICH patients than in controls (p < 0.0001). However, exclusively GFAP differed in ICH compared with IS (p < 0.0001). GFAP yielded an AUC of 0.86 for differentiating between ICH and IS within 4.5hrs of symptom onset with a sensitivity of 61% and a specificity of 96% using a cut-off of 0.34ng/ml. Higher GFAP levels were associated with stroke severity and history of prior stroke. Our results demonstrate that blood UCH-L1 and GFAP are increased early after stroke and distinct biomarker-specific release profiles are associated with stroke characteristics and type. We also confirmed the potential of GFAP as a tool for early rule-in of ICH, while UCH-L1 was not clinically useful.