Current advances in the detection of COVID-19 and evaluation of the humoral response.

The new outbreak caused by coronavirus SARS-CoV-2 started at the end of 2019 and was declared a pandemic in March 2020. Since then, several diagnostic approaches have been re-adapted, and also improved from the previous detections of SARS and MERS coronavirus. The best strategy to handle this situation seems to rely on a triad of detection methods: (i) highly sensitive and specific techniques as the gold standard method, (ii) easier and faster point of care tests accessible for large population screening, and (iii) serology assays to complement the direct detection and to use for surveillance. In this study, we assessed the techniques and tests described in the literature, their advantages and disadvantages, and the interpretation of the results. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is undoubtedly the gold standard technique utilized not only for diagnostics, but also as a standard for comparison and validation of newer approaches. Other nucleic acid amplification methods have been shown to be adequate as point of care (POC) diagnostic tests with similar performance as RT-qPCR. The analysis of seroconversion with immunotests shows the complexity of the immune response to COVID-19. The detection of anti-SARS-CoV-2 antibodies can also help to detect previously infected asymptomatic individuals with negative RT-qPCR tests. Nevertheless, more controlled serology cohort studies should be performed as soon as possible to understand the immune response to SARS-CoV-2.

Trans-membrane Fluorescence Enhancement by Carbon Dots: Ionic Interactions and Energy Transfer.

We report on trans-membrane interactions between blue-emitting carbon dots (CDs) and fluorescein. Hydrophobic CDs with a positive surface charge are embedded as-synthesized in the lipophilic sheet of the bilayer membrane of large synthetic phospholipid vesicles. The vesicles are prepared by mixing DOPC phospholipids and lipid molecules that contain anionic fluorescein attached to their hydrophilic head. Due to attractive electrostatic interactions, the CDs and fluorescein conjoin within the vesicle membrane, which leads to photoluminescence enhancement of fluorescein and facilitates trans-membrane energy transfer between the CDs and the dye.

Optical and Thermophoretic Control of Janus Nanopen Injection into Living Cells.

Devising strategies for the controlled injection of functional nanoparticles and reagents into living cells paves the way for novel applications in nanosurgery, sensing, and drug delivery. Here, we demonstrate the light-controlled guiding and injection of plasmonic Janus nanopens into living cells. The pens are made of a gold nanoparticle attached to a dielectric alumina shaft. Balancing optical and thermophoretic forces in an optical tweezer allows single Janus nanopens to be trapped and positioned on the surface of living cells. While the optical injection process involves strong heating of the plasmonic side, the temperature of the alumina stays significantly lower, thus allowing the functionalization with fluorescently labeled, single-stranded DNA and, hence, the spatially controlled injection of genetic material with an untethered nanocarrier.

Ca(2+)/calmodulin-dependent protein kinase II contributes to intracellular pH recovery from acidosis via Na(+)/H(+) exchanger activation.

The Na(+)/H(+) exchanger (NHE-1) plays a key role in pH(i) recovery from acidosis and is regulated by pH(i) and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pH(i) indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pH(i) recovery from the acid load (dpH(i)/dt)-an index of NHE-1 activity in HEPES buffer or in NaHCO(3) buffer in the presence of inhibition of anion transporters-was significantly decreased by the CaMKII inhibitors KN-93 or AIP. pH(i) recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing beta-galactosidase myocytes (dpH(i)/dt: 0.195+/-0.04 vs. 0.045+/-0.010 min(-)(1), respectively, n=8) and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1 indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703, and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.

Reconstitution reveals how myosin-VI self-organises to generate a dynamic mechanism of membrane sculpting.

One enigma in biology is the generation, sensing and maintenance of membrane curvature. Curvature-mediating proteins have been shown to induce specific membrane shapes by direct insertion and nanoscopic scaffolding, while the cytoskeletal motors exert forces indirectly through microtubule and actin networks. It remains unclear, whether the manifold direct motorprotein-lipid interactions themselves constitute another fundamental route to remodel the membrane shape. Here we show, combining super-resolution-fluorescence microscopy and membrane-reshaping nanoparticles, that curvature-dependent lipid interactions of myosin-VI on its own, remarkably remodel the membrane geometry into dynamic spatial patterns on the nano- to micrometer scale. We propose a quantitative theoretical model that explains this dynamic membrane sculpting mechanism. The emerging route of motorprotein-lipid interactions reshaping membrane morphology by a mechanism of feedback and instability opens up hitherto unexplored avenues of membrane remodelling and links cytoskeletal motors to early events in the sequence of membrane sculpting in eukaryotic cell biology.

Optical Nanoparticle Sorting Elucidates Synthesis of Plasmonic Nanotriangles.

We investigate the optical and morphological properties of gold nanoparticles grown by reducing a gold salt with Na2S. Lasers are tuned to the observed plasmon resonances, and the optical forces exerted on the nanoparticles are used to selectively print individual nanoparticles onto a substrate. This enables us to combine dark-field spectroscopy and scanning electron microscopy to compare the optical properties of single nanoparticles with their morphology. By arresting the synthesis at different times, we are able to investigate which type of nanoparticle is responsible for the respective resonances. We find that thin Au nanotriangles are the source of the observed near infrared (NIR) resonance. The initial lateral growth of these triangles causes the plasmon resonance to redshift into the NIR, whereas a subsequent thickening of the triangles and a concomitant truncation lead to a blueshift of the resonance. Furthermore, we find that the nanotriangles produced have extremely narrow line widths (187 ± 23 meV), show nearly isotropic scattering, and are stable for long periods of time. This shows their vast potential for applications such as in vivo imaging and bio(chemical) sensing. The method used here is generally applicable to other syntheses, and shows how complex nanostructures can be built up on substrates by selectively printing NPs of varying plasmonic resonances.