Perturb-map enables CRISPR genomics with spatial resolution and identifies regulators of tumor immune composition

The cellular architecture of a tumor, particularly immune composition, has a major impact on cancer outcome, and thus there is an interest in identifying genes that control the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We show >120 Pro-Codes can be imaged within a tumor, facilitating spatial mapping of 100s of cancer clones. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found that in Tgfbr2 knockout lesions, the TME was converted to a mucinous state and T-cells excluded, which was concomitant with increased TGFb expression and pathway activation, suggesting Tgfbr2 loss on lung cancer cells enhanced suppressive effects of TGFb on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient β cells

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

SKT

Data analysts often need to characterize a data stream as a first step to its further processing. Some of the initial insights to be gained include, e.g., the cardinality of the data set and its frequency distribution. Such information is typically extracted by using sketch algorithms, now widely employed to process very large data sets in manageable space and in a single pass over the data. Often, analysts need more than one parameter to characterize the stream. However, computing multiple sketches becomes expensive even when using high-end CPUs. Exploiting the increasing adoption of hardware accelerators, this paper proposes SKT , an FPGA-based accelerator that can compute several sketches along with basic statistics (average, max, min, etc.) in a single pass over the data. SKT has been designed to characterize a data set by calculating its cardinality, its second frequency moment, and its frequency distribution. The design processes data streams coming either from PCIe or TCP/IP, and it is built to fit emerging cloud service architectures, such as Microsoft's Catapult or Amazon's AQUA. The paper explores the trade-offs of designing sketch algorithms on a spatial architecture and how to combine several sketch algorithms into a single design. The empirical evaluation shows how SKT on an FPGA offers a significant performance gain over high-end, server-class CPUs.

Surfactant-guided spatial assembly of nano-architectures for molecular profiling of extracellular vesicles

The controlled assembly of nanomaterials into desired architectures presents many opportunities; however, current preparations lack spatial precision and versatility in developing complex nano-architectures. Inspired by the amphiphilic nature of surfactants, we develop a facile approach to guide nanomaterial integration – spatial organization and distribution – in metal-organic frameworks (MOFs). Named surfactant tunable spatial architecture (STAR), the technology leverages the varied interactions of surfactants with nanoparticles and MOF constituents, respectively, to direct nanoparticle arrangement while molding the growing framework. By surfactant matching, the approach achieves not only tunable and precise integration of diverse nanomaterials in different MOF structures, but also fast and aqueous synthesis, in solution and on solid substrates. Employing the approach, we develop a dual-probe STAR that comprises peripheral working probes and central reference probes to achieve differential responsiveness to biomarkers. When applied for the direct profiling of clinical ascites, STAR reveals glycosylation signatures of extracellular vesicles and differentiates cancer patient prognosis.

Spatial architecture of the immune microenvironment orchestrates tumor immunity and therapeutic response

Tumors are not only aggregates of malignant cells but also well-organized complex ecosystems. The immunological components within tumors, termed the tumor immune microenvironment (TIME), have long been shown to be strongly related to tumor development, recurrence and metastasis. However, conventional studies that underestimate the potential value of the spatial architecture of the TIME are unable to completely elucidate its complexity. As innovative high-flux and high-dimensional technologies emerge, researchers can more feasibly and accurately detect and depict the spatial architecture of the TIME. These findings have improved our understanding of the complexity and role of the TIME in tumor biology. In this review, we first epitomized some representative emerging technologies in the study of the spatial architecture of the TIME and categorized the description methods used to characterize these structures. Then, we determined the functions of the spatial architecture of the TIME in tumor biology and the effects of the gradient of extracellular nonspecific chemicals (ENSCs) on the TIME. We also discussed the potential clinical value of our understanding of the spatial architectures of the TIME, as well as current limitations and future prospects in this novel field. This review will bring spatial architectures of the TIME, an emerging dimension of tumor ecosystem research, to the attention of more researchers and promote its application in tumor research and clinical practice.

Directions for the development of information resource management in military libraries of higher education institutions in Poland on the example of the Library of the War Studies University and the Library of the Military University of Technology

The article addresses the directions of management development in military libraries of higher education institutions in Poland. These changes are noticeable in such areas as information technology, interpersonal communication, spatial architecture, and library budget. The article also presents two most important and largest libraries in the Polish Armed Forces, namely the Library of the War Studies University and the Library of the Military University of Technology. It provides a brief historical outline of the mother universities of these libraries and the beginnings of their activities. Then, the essential modern solutions implemented there and have directly contributed to their development, are analyzed.

Development of a New cryo-S(T)EM Technique Allowing Simultaneous STEM and SEM Imaging and its Application to Biological Samples

A new type of cryo-electron microscopy (cryo-S(T)EM) technique made possible by installing a new cryo-transfer holder and an anti-contamination trap on a scanning electron microscope (Hitachi SU9000) allowed simultaneous collection of both transmission (transmission electron microscopy, TEM) images and surface (scanning electron microscopy, SEM) images at -180°C. The ultimate temperatures of the cryo-transfer holder and the anti-contamination trap reached − 190°C and − 210°C, respectively, by applying a liquid nitrogen slush. The TEM images obtained by the new cryo-S(T)EM method showed quality equal or superior to that of images obtained by conventional 100 kV TEM, although the resolution did not improve at -180°C due to slight drifting of the sample stage. Cryo-S(T)EM also had the unexpected advantage of enabling observations of intracellular structures in thick frozen cells by accelerating the sublimation of ice surrounding the specimens. The spatial architecture of the cytoskeleton, poly-ribosome-chains, endoplasmic reticulum (ER), mitochondria, etc., became visible in thick frozen cells via sufficient (deep) sublimation of ice in combination with the unroofing method. In particular, it should be noted that the ER appeared as a wide and flat structure beneath the cell membrane while forming a large spatial network together with tubular ER.