Quantitative Imaging of Protein-Protein Interactions by Multiphoton Fluorescence Lifetime Imaging Microscopy using a Streak camera

Fluorescence Lifetime Imaging Microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. We review here the recent developments in multiphoton FLIM methods and also present a description of a novel multiphoton FLIM system using a streak camera that was developed in our laboratory. We provide an example of a typical application of the system in which we measure the fluorescence resonance energy transfer between a donor/acceptor pair of fluorescent proteins within a cellular specimen.Comment: Overview of FLIM techniques, StreakFLIM instrument, FRET application

Molecular Ultrasound Imaging of Junctional Adhesion Molecule A Depicts Acute Alterations in Blood Flow and Early Endothelial Dysregulation

Objective: The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations. Approach and Results: Flow-dependent endothelial dysfunction was induced in apolipoprotein E-deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A-targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (approximate to 14x [P<0.001] and approximate to 5x [P<0.001] higher than control, respectively), both peaking after 2 weeks. Subsequently, although JAM-A expression and JAM-A-targeted microbubbles binding persisted at a higher level at the partially ligated side, it completely normalized within 4 weeks at the contralateral side. Conclusions: Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A-targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state

Routine childhood immunisation during the COVID-19 pandemic in Africa: a benefit-risk analysis of health benefits versus excess risk of SARS-CoV-2 infection.

BACKGROUND: National immunisation programmes globally are at risk of suspension due to the severe health system constraints and physical distancing measures in place to mitigate the ongoing COVID-19 pandemic. We aimed to compare the health benefits of sustaining routine childhood immunisation in Africa with the risk of acquiring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through visiting routine vaccination service delivery points. METHODS: We considered a high-impact scenario and a low-impact scenario to approximate the child deaths that could be caused by immunisation coverage reductions during COVID-19 outbreaks. In the high-impact scenario, we used previously reported country-specific child mortality impact estimates of childhood immunisation for diphtheria, tetanus, pertussis, hepatitis B, Haemophilus influenzae type b, Streptococcus pneumoniae, rotavirus, measles, meningitis A, rubella, and yellow fever to approximate the future deaths averted before 5 years of age by routine childhood vaccination during a 6-month COVID-19 risk period without catch-up campaigns. In the low-impact scenario, we approximated the health benefits of sustaining routine childhood immunisation on only the child deaths averted from measles outbreaks during the COVID-19 risk period. We assumed that contact-reducing interventions flattened the outbreak curve during the COVID-19 risk period, that 60% of the population will have been infected by the end of that period, that children can be infected by either vaccinators or during transport, and that upon child infection the whole household will be infected. Country-specific household age structure estimates and age-dependent infection-fatality rates were applied to calculate the number of deaths attributable to the vaccination clinic visits. We present benefit-risk ratios for routine childhood immunisation, with 95% uncertainty intervals (UIs) from a probabilistic sensitivity analysis. FINDINGS: In the high-impact scenario, for every one excess COVID-19 death attributable to SARS-CoV-2 infections acquired during routine vaccination clinic visits, 84 (95% UI 14-267) deaths in children could be prevented by sustaining routine childhood immunisation in Africa. The benefit-risk ratio for the vaccinated children is 85 000 (4900-546 000), for their siblings (60 years) is 96 (14-307). In the low-impact scenario that approximates the health benefits to only the child deaths averted from measles outbreaks, the benefit-risk ratio to the households of vaccinated children is 3 (0-10); if the risk to only the vaccinated children is considered, the benefit-risk ratio is 3000 (182-21 000). INTERPRETATION: The deaths prevented by sustaining routine childhood immunisation in Africa outweigh the excess risk of COVID-19 deaths associated with vaccination clinic visits, especially for the vaccinated children. Routine childhood immunisation should be sustained in Africa as much as possible, while considering other factors such as logistical constraints, staff shortages, and reallocation of resources during the COVID-19 pandemic. FUNDING: Gavi, the Vaccine Alliance; Bill & Melinda Gates Foundation

Association of Vitamin B12 Deficiency with Fatigue and Depression after Lacunar Stroke

Background: In lacunar stroke patients vitamin B12 deficiency is often found and a relationship with the degree of periventricular white matter lesions (pWMLs) is suggested. Given the known relationships between WMLs and depression and between depression and fatigue after stroke, we studied both depression and fatigue in lacunar stroke patients with and without vitamin B12 deficiency. Methods: In 40 first-ever lacunar stroke patients vitamin B12 levels were determined and self-report questionnaires for fatigue and depression were completed three months after stroke. Results: Lacunar stroke patients with vitamin B12 deficiency (N = 13) reported significantly more fatigue (90.7 versus 59.4; p =.001) and depressive symptoms (6.62 versus 3.89; p,.05) than those without (N = 27). In regression analyses, vitamin B12 deficiency was significantly and independently associated with the presence of severe fatigue and clinically significant depression. Conclusions: Our preliminary results suggest a relationship between vitamin B12 deficiency and increased levels of fatigue and depression in lacunar stroke patients. If these findings could be replicated in a larger and general stroke sample, thi

Calcitonin Gene-Related Peptide Selectively Relaxes Contractile Responses to Endothelin-1 in Rat Mesenteric Resistance Arteries □ S

ABSTRACT We tested the hypothesis that endothelin-1 (ET-1) modulates sensory-motor nervous arterial relaxation by prejunctional and postjunctional mechanisms. Isolated rat mesenteric resistance arteries were investigated with immunohistochemistry, wiremyography, and pharmacological tools. ET A -and ET B -receptors could be visualized on the endothelium and smooth muscle and on periarterial fibers containing calcitonin gene-related peptide (CGRP). Arterial contractile responses to ET-1 (0.25-16 nM) were not modified by blockade of ET B -receptors, NOsynthase, and cyclooxygenase or desensitization of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) with capsaicin. ET-1 reversed relaxing responses to CGRP in depolarized arteries. This effect was inhibited by ET Aantagonists. It was not selective because ET-1 also reversed relaxing responses to Na-nitroprusside (SNP) and because phenylephrine (PHE; 0.25-16 M) similarly reversed relaxing responses to CGRP or SNP. Conversely, contractile responses to ET-1 were, compared with PHE, hypersensitive to the relaxing effects of the TRPV1-agonist capsaicin and to exogenous CGRP, but not to acetylcholine, forskolin, pinacidil, or SNP. In conclusion, ET-1 does not stimulate sensory-motor nervous arterial relaxation, but ET A -mediated arterial contractions are selectively sensitive to relaxation by the sensory neurotransmitter CGRP. This does not involve NO, cAMP, or ATP-sensitive K ϩ channels

Reliability and validity of DTI-based indirect disconnection measures

White matter connections enable the interaction within and between brain networks. Brain lesions can cause structural disconnections that disrupt networks and thereby cognitive functions supported by them. In recent years, novel methods have been developed to quantify the extent of structural disconnection after focal lesions, using tractography data from healthy controls. These methods, however, are indirect and their reliability and validity have yet to be fully established. In this study, we present our implementation of this approach, in a tool supplemented by uncertainty metrics for the predictions overall and at voxel-level. These metrics give an indication of the reliability and are used to compare predictions with direct measures from patients' diffusion tensor imaging (DTI) data in a sample of 95 first-ever stroke patients. Results show that, except for small lesions, the tool can predict fiber loss with high reliability and compares well to direct patient DTI estimates. Clinical utility of the method was demonstrated using lesion data from a subset of patients suffering from hemianopia. Both tract-based measures outperformed lesion localization in mapping visual field defects and showed a network consistent with the known anatomy of the visual system. This study offers an important contribution to the validation of structural disconnection mapping. We show that indirect measures of structural disconnection can be a reliable and valid substitute for direct estimations of fiber loss after focal lesions. Moreover, based on these results, we argue that indirect structural disconnection measures may even be preferable to lower-quality single subject diffusion MRI when based on high-quality healthy control datasets