The potential of sentence imitation tasks for assessment of language abilities in sequential bilingual children

Sentence repetition tasks are increasingly recognised as a useful clinical tool for diagnosing language impairment in children. They are quick to administer, can be carefully targeted to elicit specific sentence structures, and are particularly informative about children’s lexical and morphosyntactic knowledge. This chapter exlores the theoretical potential of sentence repetition for assessment of sequential bilingual children, and presents three studies comparing performance of sequential bilingual children with monolingual children’s performance on standardised sentence repetition tests in Hebrew (children with L1 Russian, age 5-7 years, and L1 English, age 4½-6½ years), German (children with L1 Russian, age 4-7 years) and English (children with L1 Turkish, age 6-9 years). Results differed across studies: distribution of children in the Hebrew studies was in line with monolingual norms, while the majority of children in the English-Turkish study scored in a range that would be deemed impaired for monolingual children, and performance in the German-Russian study fell between these extremes. Analyses of performance within studies revealed similar discrepancies in effects of children’s exposure to L2, with significant effects of Age of Onset in the Hebrew-Russian and Hebrew-English groups and some indication of Length of Exposure effects, but no effects of either factor in the English-Turkish group. Multiple differences between these studies preclude direct inferences about the reasons for these different results: studies differed in content, methods and scoring of sentence repetition tests, and in ages, languages, language exposure, and socioeconomic status of participants. It is possible that socioeconomic differences are associated with differences in language experience that are equally or more important than onset and length of exposure. Collectively, these studies demonstrate that sentence repetition provides a measure of children’s proficiency in their L2, but that the use of sentence repetition in clinical assessment requires caution unless norms are available for the child’s bilingual community. As a next step, it is proposed that sentence repetition tests using early-acquired vocabulary and targeting aspects of sentence structure known to be difficult for monolingual children with language impairments should be developed in different target languages. This will allow us to explore further the factors that influence attainment of basic morphosyntax in sequential bilingual children, and the point at which sentence repetition, as a measure of morphosyntax, can help to identify children requiring clinical intervention

In vitro and ex vivo strategies for intracellular delivery

Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.National Institutes of Health (U.S.) (R01GM101420-01A1

Live-cell protein labelling with nanometre precision by cell squeezing

Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (~1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy

Laser Printing of Multilayered Alternately Conducting and Insulating Microstructures

Production of multilayered microstructures composed of conducting and insulating materials is of great interest as they can be utilized as microelectronic components. Current proposed fabrication methods of these microstructures include top-down and bottom-up methods, each having their own set of drawbacks. Laser-based methods were shown to pattern various materials with micron/sub-micron resolution; however, multilayered structures demonstrating conducting/insulating/conducting properties were not yet realized. Here, we demonstrate laser printing of multilayered microstructures consisting of conducting platinum and insulating silicon oxide layers by a combination of thermally driven reactions with microbubble-assisted printing. PtCl2 dissolved in N-methyl-2-pyrrolidone (NMP) was used as a precursor to form conducting Pt layers, while tetraethyl orthosilicate dissolved in NMP formed insulating silicon oxide layers identified by Raman spectroscopy. We demonstrate control over the height of the insulating layer between ∼50 and 250 nm by varying the laser power and number of iterations. The resistivity of the silicon oxide layer at 0.5 V was 1.5 × 1011 ωm. Other materials that we studied were found to be porous and prone to cracking, rendering them irrelevant as insulators. Finally, we show how microfluidics can enhance multilayered laser microprinting by quickly switching between precursors. The concepts presented here could provide new opportunities for simple fabrication of multilayered microelectronic devices

A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk.

BackgroundTumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway.ResultTo develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior.ConclusionThese results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance

Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8[superscript +]T-cells, and not CD4[superscript +]T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8[superscript +]T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8[superscript +]T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8[superscript +]T-cells, and decoupling of antigen uptake from B-cell activation.Kathy and Curt Marble Cancer Research Fund (Frontier Research Programme Grant)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32CA180586

Plasma membrane recovery kinetics of a microfluidic intracellular delivery platform

Intracellular delivery of materials is a challenge in research and therapeutic applications. Physical methods of plasma membrane disruption have recently emerged as an approach to facilitate the delivery of a variety of macromolecules to a range of cell types. We use the microfluidic CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content of the surrounding buffer (recovery time ~5 min without calcium vs. ~30 s with calcium). Moreover, we illustrate that manipulation of the membrane repair kinetics can yield up to 5× improvement in delivery efficiency without significantly impacting cell viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells. Subsequent manipulation of membrane repair kinetics also enables the delivery of larger materials, such as antibodies, to these difficult to manipulate cells. This work provides insight into the membrane repair process in response to mechanical delivery and could potentially enable the development of improved delivery methods.National Institutes of Health (U.S.) (Grant RC1 EB011187-02)National Institutes of Health (U.S.) (Grant R01GN101420-01A1)Kathy and Curt Marble Cancer Research FundNational Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant MPP-09Call-Langer-60