Single-cell physical phenotyping of mechanically dissociated tissue biopsies for fast diagnostic assessment

Rapid and accurate histopathological diagnosis during surgery is critical for clinical decision-making. The prevalent method of intraoperative consultation pathology is time, labour and cost intensive and requires the expertise of trained pathologists. Here, we present an alternative technique for the rapid, label-free analysis of biopsy samples by sequentially assessing the physical phenotype of singularized, suspended cells in high-throughput. This new diagnostic pipeline combines enzyme-free, mechanical dissociation of tissues with real-time deformability cytometry at measurement rates of 100 – 1,000 cells/sec, and machine learning-based analysis. We show that physical phenotype parameters extracted from brightfield images of single cells can be used to distinguish subpopulations of cells in various tissues, without prior knowledge or the need for molecular markers. Further, we demonstrate the potential of our method for inflammatory bowel disease diagnostics. Using unsupervised dimensionality reduction and logistic regression, we accurately differentiate between healthy and tumorous tissue in both mouse and human biopsy samples. The method delivers results within 30 minutes, laying the groundwork for a fast and marker-free diagnostic pipeline to detect pathological changes in solid biopsies.

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

Intraoperative Resting-State Functional Connectivity Based on RGB Imaging

RGB optical imaging is a marker-free, contactless, and non-invasive technique that is able to monitor hemodynamic brain response following neuronal activation using task-based and resting-state procedures. Magnetic resonance imaging (fMRI) and functional near infra-red spectroscopy (fNIRS) resting-state procedures cannot be used intraoperatively but RGB imaging provides an ideal solution to identify resting-state networks during a neurosurgical operation. We applied resting-state methodologies to intraoperative RGB imaging and evaluated their ability to identify resting-state networks. We adapted two resting-state methodologies from fMRI for the identification of resting-state networks using intraoperative RGB imaging. Measurements were performed in 3 patients who underwent resection of lesions adjacent to motor sites. The resting-state networks were compared to the identifications provided by RGB task-based imaging and electrical brain stimulation. Intraoperative RGB resting-state networks corresponded to RGB task-based imaging (DICE:0.55±0.29). Resting state procedures showed a strong correspondence between them (DICE:0.66±0.11) and with electrical brain stimulation. RGB imaging is a relevant technique for intraoperative resting-state networks identification. Intraoperative resting-state imaging has several advantages compared to functional task-based analyses: data acquisition is shorter, less complex, and less demanding for the patients, especially for those unable to perform the tasks.