Proposal of a new method to measure FRET quantitatively in living or fixed biomedical specimens on a laser microscope

Förster Resonance Energy Transfer , abbreviated FRET , is a fluorescence phenomenon, which can be used to study and map co-localizations and dynamics of co-localizations at nanometer precision on a light microscope. FRET has been described as a spectroscopic ruler . The efficiency of the radiationless energy transfer from an excited chromophore, the donor , to another chromophore, the acceptor , the excitation energy of which approximately matches the energy to be released by the donor, is dependent on the sixth power of the mutual distance between the two molecules in space. We propose a new, non-destructive technique for measuring FRET quantitatively and at high spatial and temporal resolution on a laser scanning microscope: Two laser beams of wavelengths suitable for the mutually exclusive excitation of the donor and the acceptor, the donor beam and the acceptor beam , respectively, are intensity modulated by means of two electro optical modulators (EOM). The modulation patterns are rectangular at duty cycle ½. The modulation frequencies differ slightly. The acceptor beam is saturating the acceptor so that it cannot accept energy from the donor. The saturation is modulated in the same way as the acceptor beam. Since the donor beam also is modulated, though at a frequency slightly different from that of the acceptor beam, the intensity of the released donor fluorescence is modulated with the beat frequency of the frequencies of the two laser beam modulations and can be detected and interpreted in quantitative terms by means of a lock in amplifier. Copyright 2011 Society of Photo-Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited

The battle for COVID-19 vaccines highlights the need for a new global governance mechanism

To the Editor—Although the rapid development of several vaccines against COVID-19 is an unparalleled scientific accomplishment, one made possible through the collaboration of researchers, industry and funding bodies, the absence of a system that secures equitable access to vaccines has uncovered deep fissures in the global governance systems for health, as noted in a recent Nature Medicine Editorial. For example, advance purchase agreements for vaccines against COVID-19 have favored affluent countries, allowing them to secure 150–500% of their predicted needs, while many citizens of low-and middle-income countries (LMICs) will remain unvaccinated until 2024. Additionally, the power of patent-holders and pharmaceutical companies to place conditions on the use of vaccines prices out access for LMICs, and bilateral purchasing deals are rarely disclosed. By affording priority on the basis of economic or political power, today’s discourse clearly deviates from previous ethical and public-health principles of maximizing lives or life-years saved, and the sentiment that “people’s entitlement to lifesaving resources should not depend on nationality”. The COVID-19 pandemic has tested wealthy nations’ commitments to Agenda 2030 and to ‘leaving no one behind’ at the same time that it has revealed democratic deficits, institutional rigidity, weak accountability systems, and inadequate policy space that protects health-governance systems from economic goals. Thus, the as-yet-limited support for the vaccine-sharing and allocation principles of the COVAX initiative may be a sign not only of a moral catastrophe, to quote the director-general of the World Health Organization (WHO), but also of inadequate global accountability mechanisms that exposes the consequences of commercial determinants of health

A prize for global health in the name of Rudolf Virchow

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Local impact of perivascular plaques on cerebral blood flow dynamics in a transgenic mouse model of Alzheimer's disease.

Cerebrovascular pathology is closely coupled to cognitive function decline, as indicated by numerous studies at the system level. To better understand the mechanisms of this cognitive decline it is important to resolve how pathological changes in the vasculature - such as perivascular plaques - affect local cerebral blood flow dynamics. This issue is ideally studied in the intact brain at very high spatial resolution. Here, we describe initial results obtained by an approach based on in vivo observation by multi-photon microscopy of vascular plaques and local blood flow measurements in a transgenic mouse model engineered to express the human amyloid precursor protein with the Swedish and Arctic mutations. These mice exhibit a striking abundance of perivascular plaques in the cerebral cortex and are well suited to investigate vascular pathology in Alzheimer's disease

Specialized membrane domains for water transport in glial cells: high resolution immunogold cytochemistry of aquaporin-4 in rat brain

Membrane water transport is critically involved in brain volume homeostasis and in the pathogenesis of brain edema. The cDNA encoding aquaporin-4 (AQP4) water channel protein was recently isolated from rat brain. We used immunocytochemistry and high-resolution immunogold electron microscopy to identify the cells and membrane domains that mediate water flux through AQP4. The AQP4 protein is abundant in glial cells bordering the subarachnoidal space, ventricles, and blood vessels. AQP4 is also abundant in osmosensory areas, including the supraoptic nucleus and subfornical organ. Immunogold analysis demonstrated that AQP4 is restricted to glial membranes and to subpopulations of ependymal cells. AQP4 is particularly strongly expressed in glial membranes that are in direct contact with capillaries and pia. The highly polarized AQP4 expression indicates that these cells are equipped with specific membrane domains that are specialized for water transport, thereby mediating the flow of water between glial cells and the cavities filled with CSF and the intravascular space

Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders

Mixed Electrical–Chemical Synapses in Adult Rat Hippocampus are Primarily Glutamatergic and Coupled by Connexin-36

Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for “mixed” (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into weakly fixed CA3pyr was detected in MF axons that contacted four injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold labeling revealed diverse sizes and morphologies of connexin-36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328–1140 connexons), three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin-section images of a CA3pyr, but none were found by immunogold labeling, suggesting the rarity of GABAergic mixed synapses. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal neurons

A call for an immediate ceasefire and peaceful end to the Russian aggression against Ukraine

The Lancet–SIGHT Commission condemns the Russian Government's aggression against Ukraine and its attacks on civilians and civilian infrastructure, including health workers and hospitals. We support the March 2, 2022 UN General Assembly (UNGA) resolution ES-11/1 that “deplores in the strongest terms the aggression by the Russian Federation”. The indiscriminate use of weaponry violates international humanitarian law and has caused catastrophic health impacts, especially on children, older people, and disabled persons, and social and economic disruptions that will be long lasting. There are nuclear risks, both from Russian attacks on Ukrainian nuclear facilities and the potential for nuclear weapons use. There is a further risk that Russia might use chemical or biological weapons. We call for an immediate ceasefire and the appointment of a mediator to facilitate negotiations for a sustainable and peaceful settlement on the basis of international law to end the conflict. We urge the global health community to deliver humanitarian assistance impartially to all those affected by and fleeing the war; document atrocities committed against civilians and the devastating impacts of the war; counter disinformation about the conflict; and advocate for a peaceful settlement. We also call for an end to the repression of those in Russia protesting the war.http://www.thelancet.comhj2023Economic