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"Ferrante, C."
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Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro
Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower 'vascular' channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis.
Journal Article
Coherent anti-Stokes Raman spectroscopy of single and multi-layer graphene
Spontaneous Raman spectroscopy is a powerful characterization tool for graphene research. Its extension to the coherent regime, despite the large nonlinear third-order susceptibility of graphene, has so far proven challenging. Due to its gapless nature, several interfering electronic and phononic transitions concur to generate its optical response, preventing to retrieve spectral profiles analogous to those of spontaneous Raman. Here we report stimulated Raman spectroscopy of the G-phonon in single and multi-layer graphene, through coherent anti-Stokes Raman Scattering. The nonlinear signal is dominated by a vibrationally non-resonant background, obscuring the Raman lineshape. We demonstrate that the vibrationally resonant coherent anti-Stokes Raman Scattering peak can be measured by reducing the temporal overlap of the laser excitation pulses, suppressing the vibrationally non-resonant background. We model the spectra, taking into account the electronically resonant nature of both. We show how coherent anti-Stokes Raman Scattering can be used for graphene imaging with vibrational sensitivity.
Coherent anti-Stokes Raman Scattering (CARS) accesses the vibrational properties of a material via nonlinear four-wave mixing (FWM); CARS in graphene has not been observed to date despite its high nonlinear third-order susceptibility. Here, the authors devised a FWM scheme to perform stimulated Raman spectroscopy in single and multi-layer graphene through CARS.
Journal Article
Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues
Lee
et al
. provide their protocol for fluorescent
in situ
sequencing (FISSEQ) of RNA. This technique allows spatial and quantitative information about mRNA expression to be obtained simultaneously.
RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast,
in situ
hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression
in situ
in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.
Journal Article
Raman spectroscopy of graphene under ultrafast laser excitation
The equilibrium optical phonons of graphene are well characterized in terms of anharmonicity and electron–phonon interactions; however, their non-equilibrium properties in the presence of hot charge carriers are still not fully explored. Here we study the Raman spectrum of graphene under ultrafast laser excitation with 3 ps pulses, which trade off between impulsive stimulation and spectral resolution. We localize energy into hot carriers, generating non-equilibrium temperatures in the ~1700–3100 K range, far exceeding that of the phonon bath, while simultaneously detecting the Raman response. The linewidths of both G and 2D peaks show an increase as function of the electronic temperature. We explain this as a result of the Dirac cones’ broadening and electron–phonon scattering in the highly excited transient regime, important for the emerging field of graphene-based photonics and optoelectronics.
Non-equilibrium ultrafast processes in graphene entail relaxation pathways involving electron–electron and electron–phonon scattering events. Here, the authors probe graphene optical phonons at high electronic temperatures by means of Raman spectroscopy under pulsed excitation
Journal Article
Highly Multiplexed Subcellular RNA Sequencing in Situ
Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.
Journal Article
Deconstructing transcriptional heterogeneity in pluripotent stem cells
Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates; however, the regulatory circuits specifying these states and enabling transitions between them are not well understood. Here we set out to characterize transcriptional heterogeneity in mouse PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signalling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signalling pathways and chromatin regulators. Notably, either removal of mature microRNAs or pharmacological blockage of signalling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the
Myc
/
Lin28
/let-7 axis. These data provide insight into the nature of transcriptional heterogeneity in PSCs.
This study uses single-cell expression profiling of pluripotent stem cells after various perturbations, and uncovers a high degree of variability that can be inherited through cell divisions—modulating microRNA or external signalling pathways induces a ground state with reduced gene expression heterogeneity and a distinct chromatin profile.
Gene expression variation in pluripotency
Although it is recognized that pluripotent stem cells switch dynamically between distinct substates, the gene regulatory networks specifying the states and governing transitions between them are not well defined. Using single-cell expression profiling of mouse pluripotent stem cells subjected to chemical and genetic perturbations, George Daley and colleagues establish how transcriptional networks are dynamically reconfigured to drive distinct states of pluripotency. They observe a high degree of variability that can be inherited through cell divisions and find that modulating microRNA or external signalling pathways lowers the heterogeneity in gene expression and induces a distinct epigenetic state.
Journal Article
Direct observation of subpicosecond vibrational dynamics in photoexcited myoglobin
Determining the initial pathway for ultrafast energy redistribution within biomolecules is a challenge, and haem proteins, for which energy can be deposited locally in the haem moiety using short light pulses, are suitable model systems to address this issue. However, data acquired using existing experimental techniques that fail to combine sufficient structural sensitivity with adequate time resolution have resulted in alternative hypotheses concerning the interplay between energy flow among highly excited vibrational levels and potential concomitant electronic processes. By developing a femtosecond-stimulated Raman set-up, endowed with the necessary tunability to take advantage of different resonance conditions, here we visualize the temporal evolution of energy redistribution over different vibrational modes in myoglobin. We establish that the vibrational energy initially stored in the highly excited Franck–Condon manifold is transferred with different timescales into low- and high-frequency modes, prior to slow dissipation through the protein. These findings demonstrate that a newly proposed mechanism involving the population dynamics of specific vibrational modes settles the controversy on the existence of transient electronic intermediates.
Difficulties in experimentally achieving simultaneous structural sensitivity and time resolution have hindered the real-time mapping of the vibrational energy relaxation pathways in biomacromolecules. Now, using ultrashort light pulses to locally deposit excess energy in a protein-bound haem, the temporal evolution of the subsequent energy flow has been monitored, unravelling vibrational couplings that lead to mode-specific temperature changes.
Journal Article
Probing ultrafast photo-induced dynamics of the exchange energy in a Heisenberg antiferromagnet
Femtosecond stimulated Raman experiments on the antiferromagnetic system KNiF
3
are implemented to understand how the exchange interaction — a crucial interaction that rules magnetic phenomena — is influenced by ultrafast optical excitation.
Manipulating the macroscopic phases of solids using ultrashort light pulses has resulted in spectacular phenomena, including metal–insulator transitions
1
,
2
,
3
, superconductivity
4
and subpicosecond modification of magnetic order
5
. The development of this research area strongly depends on the understanding and optical control of fundamental interactions in condensed matter, in particular the exchange interaction. However, disentangling the timescales relevant for the contributions of the exchange interaction and spin dynamics to the exchange energy,
E
ex
, is a challenge. Here, we introduce femtosecond stimulated Raman scattering to unravel the ultrafast photo-induced dynamics of magnetic excitations at the edge of the Brillouin zone. We find that femtosecond laser excitation of the antiferromagnet KNiF
3
triggers a spectral shift of the two-magnon line, the energy of which is proportional to
E
ex
. By unravelling the photo-induced modification of the two-magnon line frequency from a dominating nonlinear optical effect, we find that
E
ex
is increased by the electromagnetic stimulus.
Journal Article
Ectopic Lymphoid Follicle Formation and Human Seasonal Influenza Vaccination Responses Recapitulated in an Organ‐on‐a‐Chip
Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non‐human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B‐ and T‐lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two‐channel organ‐on‐a‐chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center‐like LFs contain B cells expressing Activation‐Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte‐derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene‐in‐water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti‐hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales. Primary human lymphocytes taken from blood are reprogrammed into lymphoid follicles (LF) by superfusion in an organ chip device. These ectopic LFs express AID and can be induced to form plasma cells and antigen‐specific antibodies upon activation with different stimuli including adjuvants and seasonal influenza vaccines.
Journal Article