Theory Theory of Coherent Raman Scattering, Eric Olaf Potma and Shaul MukamelCoherent Raman Scattering under Tightly Focused Conditions, Eric Olaf Potma, Xiaoliang Sunney Xie, Andreas Volkmer, and ...Ji-Xin ChengPlatformsConstruction of a Coherent Raman Microscope, Brian G. Sarr and Xiaoliang Sunney XieStimulated Raman Scattering Microscopy, Christian Freudiger and Xiaoliang Sunney XieFemtosecond versus Picosecond Pulses for Coherent Raman Microscopy, Mikhail N. Slipchenko, Delong Zhang, and Ji-Xin ChengWide-Field CARS Microscopy, Alexander Jesacher, Gregor Thalhammer, Stefan Bernet, and Monika Ritsch-MarteVibrational Spectromicroscopy by Coupling Coherent Raman Imaging with Spontaneous Raman Spectral Analysis, Mikhail N. Slipchenko and Ji-Xin ChengCoherent Control in CARS, Jonathan M. Levitt, Ori Katz, and Yaron SilberbergFourier Transform CARS Microscopy, Jennifer P. OgilvieCRS with Alternative Beam Profiles, Varun Raghunathan, Hyunmin Kim, Stephan Stranick, and Eric Olaf PotmaVibrational Phase Microscopy, Martin Jurna, Cees Otto, and Herman L. OfferhausMultiplex CARS Microscopy, James P.R. Day, Katrin F. Domke, Gianluca Rago, Erik M. Vartiainen, and Mischa BonnInterferometric Multiplex CARS, Sang-Hyun LimPhotonic Crystal Fiber-Based Broadband CARS Microscopy, Marcus T. Cicerone, Young Jong Lee, Sapun H. Parekh, and Khaled A. AamerMultiplex Stimulated Raman Scattering Microscopy, Dan Fu and Xiaoliang Sunney XieApplicationsImaging Myelin Sheath Ex Vivo and In Vivo by CARS Microscopy, Yan Fu, Yunzhou (Sophia) Shi, and Ji-Xin ChengImaging Lipid Metabolism in Caenorhabditis elegans and Other Model Organisms, Helen Fink, Christian Brackmann, and Annika EnejderLipid-Droplet Biology and Obesity-Related Health Risks, Thuc T. LeWhite Matter Injury: Cellular-Level Myelin Damage Quantification in Live Animals, Erik Bélanger, F.P. Henry, R. Vallée, M.A.
Randolph, I.E. Kochevar, J.M. Winograd, Charles P. Lin, and Daniel CôtéCARS Microscopy Study of Liquid Crystals, Heung-Shik Park and Oleg D. LavrentovichLive Cell Imaging by Multiplex CARS Microspectroscopy, Hideaki Kano Coherent Raman Scattering Imaging of Drug Delivery Systems, Ling Tong and Ji-Xin ChengApplications of Stimulated Raman Scattering Microscopy, Christian Freudiger, Daniel A. Orringer, and Xiaoliang Sunney XieApplications of Coherent Anti-Stokes Raman Spectroscopy Imaging to Cardiovascular Diseases, Han-Wei Wang, Michael Sturek, and Ji-Xin ChengApplications of CARS Microscopy to Tissue Engineering, Annika Enejder and Christian BrackmannDietary Fat Absorption Visualized by CARS Microscopy, Kimberly K. BuhmanIndex.
Natural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to ...antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under β-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance.
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•Persisters accumulate fewer antibiotics as a direct result of increased efflux rate•Persisters show higher expression of efflux-associated genes•High expression of tolC is critical to promote persister formation•Persisters combine active efflux and passive dormancy to survive antibiotic attack
By using in vivo fluorescent imaging and next-generation sequencing, Pu et al. demonstrate that bacterial persisters exhibit enhanced efflux activity in addition to physiological quiescence, a double insurance strategy to ensure its survival under antibiotic attack.
The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here ...we use high-throughput yeast display screening
to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A-F)-a grouping that is highly concordant with knowledge-based structural classifications
. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A-D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)
and group F (for example, CR3022)
, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.
We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification ...(MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.
Abstract
Different regions of the mammalian nose smell different odors. In the mouse olfactory system, spatially regulated expression of >1000 olfactory receptors (ORs) along the ...dorsomedial-ventrolateral (DV) axis forms a topological map in the main olfactory epithelium (MOE). However, the locations of most ORs along the DV axis are currently unknown. By sequencing mRNA of 12 isolated MOE pieces, we mapped out the DV locations-as quantified by "zone indices" on a scale of 1-5-of 1033 OR genes with an estimated error of 0.3 zone indices. Our map covered 81% of all intact OR genes and 99.4% of the total OR mRNA abundance. Spatial regulation tended to vary gradually along chromosomes. We further identified putative non-OR genes that may exhibit spatial expression along the DV axis.
The SARS-CoV-2 Omicron variant with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the spike (S) from Omicron reveals amino acid substitutions forging ...interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge, and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members, as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies, sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.
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•Omicron spike stably maintains an active conformation for receptor recognition•Improved stability of Omicron enhances attachment but compromises viral fusion•Mutations perturb the conformation of antigenic sites recognized by most antibodies•Structurally restrained regions of RBM can be targets for COVID-19 countermeasures
Mutations in the SARS-CoV-2 Omicron variant improve spike trimer stability that supports viral attachment but appears to compromise viral fusion. They also perturb the confirmation of antigenic sites for antibody recognition, which may contribute to immune evasion.
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages BA.2.12.1, BA.4 and BA.5 exhibit higher transmissibility than the BA.2 lineage
1
. The receptor binding and ...immune-evasion capability of these recently emerged variants require immediate investigation. Here, coupled with structural comparisons of the spike proteins, we show that BA.2.12.1, BA.4 and BA.5 (BA.4 and BA.5 are hereafter referred collectively to as BA.4/BA.5) exhibit similar binding affinities to BA.2 for the angiotensin-converting enzyme 2 (ACE2) receptor. Of note, BA.2.12.1 and BA.4/BA.5 display increased evasion of neutralizing antibodies compared with BA.2 against plasma from triple-vaccinated individuals or from individuals who developed a BA.1 infection after vaccination. To delineate the underlying antibody-evasion mechanism, we determined the escape mutation profiles
2
, epitope distribution
3
and Omicron-neutralization efficiency of 1,640 neutralizing antibodies directed against the receptor-binding domain of the viral spike protein, including 614 antibodies isolated from people who had recovered from BA.1 infection. BA.1 infection after vaccination predominantly recalls humoral immune memory directed against ancestral (hereafter referred to as wild-type (WT)) SARS-CoV-2 spike protein. The resulting elicited antibodies could neutralize both WT SARS-CoV-2 and BA.1 and are enriched on epitopes on spike that do not bind ACE2. However, most of these cross-reactive neutralizing antibodies are evaded by spike mutants L452Q, L452R and F486V. BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1. Nevertheless, these neutralizing antibodies are largely evaded by BA.2 and BA.4/BA.5 owing to D405N and F486V mutations, and react weakly to pre-Omicron variants, exhibiting narrow neutralization breadths. The therapeutic neutralizing antibodies bebtelovimab
4
and cilgavimab
5
can effectively neutralize BA.2.12.1 and BA.4/BA.5, whereas the S371F, D405N and R408S mutations undermine most broadly sarbecovirus-neutralizing antibodies. Together, our results indicate that Omicron may evolve mutations to evade the humoral immunity elicited by BA.1 infection, suggesting that BA.1-derived vaccine boosters may not achieve broad-spectrum protection against new Omicron variants.
xie discusses how advances in single-molecule imaging and manipulation have changed the way many biological problems are addressed and have generated new knowledge in the field. Since the 1990s, ...advances in single-molecule imaging and manipulation have led to the emergence of single-molecule biology (ie, the probing and understanding of biological behaviors on a single-molecule basis). Due to contributions from many laboratories around the world, this has changed the way many biological problems are addressed and generated much new knowledge. One early example of single-molecule biology is the study of turnovers of the enzyme cholesterol oxidase. The enzyme contains a flavin moiety that is naturally fluorescent in its oxidized form, but not in its reduced form.
SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 ...variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242-244 deletion (242-244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242-244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.
Raman microscopy is a quantitative, label-free, and noninvasive optical imaging technique for studying inhomogeneous systems. However, the feebleness of Raman scattering significantly limits the use ...of Raman microscopy to low time resolutions and primarily static samples. Recent developments in narrowband stimulated Raman scattering (SRS) microscopy have significantly increased the acquisition speed of Raman based label-free imaging by a few orders of magnitude, at the expense of reduced spectroscopic information. On the basis of a spectral focusing approach, we present a fast SRS hyperspectral imaging system using chirped femtosecond lasers to achieve rapid Raman spectra acquisition while retaining the full speed and image quality of narrowband SRS imaging. We demonstrate that quantitative concentration determination of cholesterol in the presence of interfering chemical species can be achieved with sensitivity down to 4 mM. For imaging purposes, hyperspectral imaging data in the C–H stretching region is obtained within a minute. We show that mammalian cell SRS hyperspectral imaging reveals the spatially inhomogeneous distribution of saturated lipids, unsaturated lipids, cholesterol, and protein. The combination of fast spectroscopy and label-free chemical imaging will enable new applications in studying biological systems and material systems.
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