Search

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

Many microtubule (MT) functions are mediated by a diverse class of proteins (+TIPs) at growing MT plus ends that control intracellular MT interactions and dynamics and depend on end-binding proteins (EBs) [1]. Cryoelectron microscopy has recently identified the EB binding site as the interface of four tubulin dimers that undergoes a conformational change in response to β-tubulin GTP hydrolysis [2, 3]. Doublecortin (DCX), a MT-associated protein (MAP) required for neuronal migration during cortical development [4, 5], binds to the same site as EBs [6], and recent in vitro studies proposed DCX localization to growing MT ends independent of EBs [7]. Because this conflicts with observations in neurons [8, 9] and the molecular function of DCX is not well understood, we revisited intracellular DCX dynamics at low expression levels. Here, we report that DCX is not a +TIP in cells but, on the contrary, is excluded from the EB1 domain. In addition, we find that DCX-MT interactions are highly sensitive to MT geometry. In cells, DCX binding was greatly reduced at MT segments with high local curvature. Remarkably, this geometry-dependent binding to MTs was completely reversed in the presence of taxanes, which reconciles incompatible observations in cells [9] and in vitro [10]. We propose a model explaining DCX specificity for different MT geometries based on structural changes induced by GTP hydrolysis that decreases the spacing between adjacent tubulin dimers [11]. Our data are consistent with a unique mode of MT interaction in which DCX specifically recognizes this compacted GDP-like MT lattice.

Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. We describe a system for inducing the formation of focal adhesions in normally non-ECM-adherent, nonmotile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a nonpolarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of nonmotile, nonpolarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNA interference (RNAi) depletion of myosin II and focal adhesion kinase, suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.

Simple Summary: Patients are the center and focus of care delivery, but frequently, their perceptions are overlooked. Patients are vital to promoting optimal oncology nursing care and should be considered in order to provide patient-centered holistic care. The Quality of Oncology Nursing Care Scale was translated and validated cross-culturally for the Portuguese context. In Portugal, there is no instrument that evaluates the quality of nursing care provided to cancer patients, so our study fills this major gap in the scientific evidence. This scale should be used by nurses, nurse managers, and researchers that strive to improve patient, nurse, and organizational outcomes. Background: Quality assessment in oncology nursing care has been a growing topic in the literature, gaining relevance as oncological nursing care becomes more complex as the science progresses. However, there are no instruments that assess the perception of the quality of oncology nursing care from the point of view of patients for the Portuguese population. Thus, the cross-cultural translation and validation of the Quality of Oncology Nursing Care Scale (QONCS) was performed for the Portuguese context. This instrument allows nurses to assess patients' self-perception of the quality of nursing care provided in an oncological setting. It also allows researchers to compare the results obtained internationally with the application of this scale. Methods: This is a methodological study, with two distinct phases: the first corresponded to the translation and cultural adaptation of the scale to the Portuguese context, and the second consisted of the psychometric validation of the QONCS, which included factor analysis and the evaluation of the psychometric properties of the instrument. We obtained responses from 402 patients from a Portuguese oncology hospital. Results: The Portuguese version of the Quality of Oncology Nursing Care Scale (QONCS_PT) consists of 34 items inserted into a tetra-factorial model, which explains a total variance of the instrument of 69.8%. A Cronbach's alpha of 0.93 was obtained for the complete instrument. Conclusions: QONCS_PT has a competent and reliable structure. The scale's validity was assured and can be used in the Portuguese population, as it is useful for direct care provision but also for researchers and managers. [ABSTRACT FROM AUTHOR]

New nickel and copper bisdithiolene complexes were prepared as tetraphenylphosphonium (Ph4P+) salts, (Ph4P)n[M(3cbdt)2], [n = 1, 2; M = Ni, Cu; 3cbdt = 3-cyanobenzene-1,2-dithiolate]: (Ph4P)2[Ni(3cbdt)2] (1); Ph4P[Ni(3cbdt)2] (2); (Ph4P)2[Cu(3cbdt)2] (3); Ph4P[Cu(3cbdt)2] (4), and characterised by single crystal X-ray diffraction, cyclic voltammetry, EPR and static magnetic susceptibility for the paramagnetic species. These Cu and Ni complexes obtained as stable monoanionic and dianionic anions are the first members of this family of cyano benzene functionalized bisdithiolene transition metal complexes, opening the way to the preparation of analogous compounds with other transition metals and their future use as building blocks for the preparation of molecular materials. Their properties are discussed in comparison with the corresponding complexes based on the 4-cyanobenzene-1,2-dithiolate (4cbdt) ligand previously described by us. The salts (Ph4P)n[M(3cbdt)2], [n = 1, 2; M = Ni, Cu] present the metal complexes always in a square-planar coordination geometry with a ligand trans configuration, but revealing a rich polymorphism. The Ni and Cu compounds are isostructural and occur as two polymorphs (α, β) with the exception of the salt of the monoanionic Ni complex, that was found only as the α-polymorph. The salt of the dianionic Ni complex was found to crystallise also with one acetonitrile molecule, when recrystallised from this solvent. The salts of monoanionic [Ni(3cbdt)2] and dianionic [Cu(3cbdt)2] complexes have been characterised by EPR and static magnetic susceptibility as paramagnetic S = 1/2 species. [ABSTRACT FROM AUTHOR]

Aims Isolation and characterization of lactobacilli from human milk and determination of their probiotic, technological, and in vitro health-promoting properties with a view to their potential use in food fermentation. Methods and Results Seven lactobacilli isolates were obtained from human milk and identified as Lacticaseibacillus paracasei (isolates BM1–BM6) and Lactobacillus gasseri (BM7). The isolates were examined in vitro for their technological, probiotic, and health-promoting potential. Overall, all isolates showed important technological properties based on the ability to grow in milk whey, a high to moderate acidification capacity and the absence of undesirable enzymatic activities. Lacticaseibacillus gasseri (BM7) differed from the L. paracasei isolates by the absence of several glycosidases and the inability to ferment lactose. Isolates L. paracasei BM3 and BM5 produced exopolysaccharides (EPS) from lactose. All isolates showed probiotic potential as they were tolerant to simulated gastrointestinal conditions, had high cell surface hydrophobicity, had not acquired resistance to relevant antibiotics and had no virulence characteristics. All L. paracasei showed high antimicrobial activity against various pathogenic bacteria and fungi, while L. gasseri showed a narrower spectrum of antimicrobial activity. All isolates showed health-promoting potential in vitro , as evidenced by high cholesterol-lowering activity, high ACE inhibitory activity and marked antioxidant activity. Conclusions All strains showed excellent probiotic and technological properties for use in lactic ferments. [ABSTRACT FROM AUTHOR]