Hyperactive Rac stimulates cannibalism of living target cells and enhances CAR-M-mediated cancer cell killing

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy

Deep Learning in Phosphoproteomics: Methods and Application in Cancer Drug Discovery

Protein phosphorylation is a key post-translational modification (PTM) that is a central regulatory mechanism of many cellular signaling pathways. Several protein kinases and phosphatases precisely control this biochemical process. Defects in the functions of these proteins have been implicated in many diseases, including cancer. Mass spectrometry (MS)-based analysis of biological samples provides in-depth coverage of phosphoproteome. A large amount of MS data available in public repositories has unveiled big data in the field of phosphoproteomics. To address the challenges associated with handling large data and expanding confidence in phosphorylation site prediction, the development of many computational algorithms and machine learning-based approaches have gained momentum in recent years. Together, the emergence of experimental methods with high resolution and sensitivity and data mining algorithms has provided robust analytical platforms for quantitative proteomics. In this review, we compile a comprehensive collection of bioinformatic resources used for the prediction of phosphorylation sites, and their potential therapeutic applications in the context of cancer

Immunomodulation, Toxicity, and Therapeutic Potential of Nanoparticles

Altered immune responses associated with human disease conditions, such as inflammatory and infectious diseases, cancers, and autoimmune diseases, are among the primary causes of morbidity across the world. A wealth of studies has demonstrated the efficiency of nanoparticles (NPs)-based immunotherapy strategies in different laboratory model systems. Nanoscale dimensions (<100 nm) enable NPs to have increased surface area to volume ratio, surface charge, and reactivity. Physicochemical properties along with the shapes, sizes, and elasticity influence the immunomodulatory response induced by NPs. In recent years, NPs-based immunotherapy strategies have attained significant focus in the context of cancers and autoimmune diseases. This rapidly growing field of nanomedicine has already introduced ~50 nanotherapeutics in clinical practices. Parallel to wide industrial applications of NPs, studies have raised concerns about their potential threat to the environment and human health. In past decades, a wealth of in vivo and in vitro studies has demonstrated the immunotoxicity potential of various NPs. Given that the number of engineered/designed NPs in biomedical applications is continuing to increase, it is pertinent to establish the toxicity profile for their safe and intelligent use in biomedical applications. The review is intended to summarize the NPs-induced immunomodulation pertaining to toxicity and therapeutic development in human health

Investigating the Effectiveness of Plant-Mediated Cerium Oxide Nanoparticles as Larvicidal Agents against the Dengue Vector <i>Aedes aegypti</i>

Aedes aegypti mosquito is responsible for the transmission of some of the most serious vector-borne diseases affecting humans, including dengue fever, chikungunya, and Zika. The only effective method for minimizing their transmission is vector control. In this work, an environmentally friendly method for synthesizing cerium oxide nanoparticles (CeO2 NPs) is highlighted, and the larvicidal activity against Ae. aegypti was studied. This method uses the aqueous extract of Bruguiera cylindrica leaves (BL) as an oxidizer and stabilizing agent. UV–Vis spectroscopy presented a distinctive absorbance band at 303 nm for CeO2 NPs with a band gap of 3.17 eV. The functional groups from the plant extract connected to CeO2 NPs were identified by FT-IR analysis, while X-ray diffraction revealed the cubic fluorite orientation of CeO2 NPs. Zeta potential revealed a surface charge of −20.7 mV on NPs. The formation of CeO2 NPs was confirmed by an energy dispersive spectral analysis, and TEM and DLS revealed an average diameter of 40–60 nm. The LC50 of synthesized CeO2 against Ae. aegypti fourth instar larvae was reported to be 46.28 μg/mL in 24 h. Acetylcholinesterase (47%) and glutathione S-transferase (13.51%) activity were significantly decreased in Ae. aegypti larvae exposed to synthesized CeO2 NPs versus the control larvae. All these findings propose the potential for using B. cylindrica leaves-synthesized CeO2 NPs as an efficient substitute for insecticides in the management of mosquitoes

Coordination of protrusion dynamics within and between collectively migrating border cells by myosin II.

Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo

The <i>Drosophila</i> Importin-α3 Is Required for Nuclear Import of Notch In Vivo and It Displays Synergistic Effects with Notch Receptor on Cell Proliferation

<div><p>The Notch signaling pathway controls diverse cell-fate specification events throughout development. The versatility of this pathway to influence different aspects of development comes from its multiple levels of regulation. Upon ligand-induced Notch activation, the Notch intracellular domain (Notch-ICD) is released from the membrane and translocates to the nucleus, where it transduces Notch signals by regulating the transcription of downstream target genes. But the exact mechanism of translocation of Notch-ICD into the nucleus is not clear. Here, we implicate Importin-α3 (also known as karyopherin-α3) in the nuclear translocation of Notch-ICD in <i>Drosophila</i>. Our present analyses reveal that Importin-α3 can directly bind to Notch-ICD and loss of Importin-α3 function results in cytoplasmic accumulation of the Notch receptor. Using MARCM (Mosaic Analysis with a Repressible Cell Marker) technique, we demonstrate that Importin-α3 is required for nuclear localization of Notch-ICD. These results reveal that the nuclear transport of Notch-ICD is mediated by the canonical Importin-α3/Importin-β transport pathway. In addition, co-expression of both Notch-ICD and Importin-α3 displays synergistic effects on cell proliferation. Taken together, our results suggest that Importin-α3 mediated nuclear import of Notch-ICD may play important role in regulation of Notch signaling.</p></div

Loss-of-function and gain-of-function effects of <i>imp-α3</i> on localization of endogenous Notch protein.

<p>(A1–C4) <i>imp-α3</i> mutant cells have elevated Notch protein levels. Levels of Notch (N) protein in third instar larval salivary glands (A1–B4) and eye-antennal discs (C1–C4) that contain <i>imp-α3</i> mutant clones marked by the absence of green fluorescent protein (GFP). Images in A4, B4, and C4 are merges of those in A1–A3, B1–B3, and C1–C3, respectively. High magnification of <i>imp-α3</i> clones in A1–A4 are shown in B1–B4. Note the increased levels of Notch in <i>imp-α3</i> mutant cells (arrowheads). Scale bars, 50 µm (A1–A4), 10 µm (B1–C4). (D1–D3) Ectopic expression of Importin-α3 results in the formation of cytoplasmic aggregates of endogenous Notch protein. <i>UAS-HA-imp-α3</i> transgene was expressed under the control of <i>en-GAL4</i> driver, which is expressed in posterior compartment cells of wing discs. Note that more number of Notch aggregates in cytoplasm of posterior compartment cells compare to anterior compartment cells in wing disc. Image in D3 is merge of those in D1 and D2. Inset in D3 shows higher magnification image of a single cell from posterior compartment showing many Notch aggregates. Scale bar, 5 µm.</p

Loss of <b><i>imp-α3</i></b> blocks the nuclear import of Notch-ICD.

<p>(A1–D3) MARCM-derived <i>imp-α3</i> mutant clones (A1–B3) and wild-type clones (C1–D3) in larval brain marked with green fluorescent protein (GFP). Images in A3, B3, C3, and D3 are merges of those in A1–A2, B1–B2, C1–C2, and D1–D2, respectively. High magnification of part (marked with open rectangle in A3 and C3) of GFP-marked <i>imp-α3</i> clones in A1–A3 are shown in B1–B3 and GFP-marked wild-type clones in C1–C3 are shown in D1–D3. Note the cytoplasmic localization of Notch-ICD in <i>imp-α3</i> mutant cells (arrowhead in B3), which is readily detectable in the nucleus in wild-type clonal cells as shown in D3 (arrowhead). Scale bars, 100 µm (A1–A3 and C1–C3), 10 µm (B1–B3 and D1–D3).</p

Importin-α3 displays synergistic effect with activated Notch on signaling activity of the Notch receptor.

<p>(A1–C5) Eye-antennal discs in which <i>UAS-HA-imp-α3</i> was overexpressed by <i>ey-GAL4</i> driver (A1–A4), eye-antennal discs in which <i>UAS-Notch-ICD</i> was overexpressed using <i>ey-GAL4</i> driver (B1–B4), and eye-antennal discs from individuals in which both <i>UAS-Notch-ICD</i> and <i>UAS-HA-imp-α3</i> were overexpressed by <i>ey-GAL4</i> strain (C1–C4) showing Elav expression. Note that Elav staining of larval eye discs in which <i>Notch-ICD</i> and <i>imp-α3</i> were both overexpressed showed enhanced defects in ommatidial spacing and misrotated ommatidia. Images in A3, B3, and C3 are merges of those in A1 and A2, B1 and B2, and C1 and C2, respectively. Images in A4, B4, and C4 are high magnification images of Elav expressing cells shown in A2, B2, and C2, respectively. Scale bars for A1–A3, B1–B3, and C1–C3, 50 µm and for A4, B4, and C4, 5 µm. (A5, B5, and C5) Nail polish imprints of adult eyes of genotypes as in A1–A4, B1–B4, and C1–C4, respectively. Note the co-expression of <i>Notch-ICD</i> and <i>imp-α3</i> results in a considerable enhancement of the adult eye phenotype with more frequent fusion of ommatidia and appearance of abnormally sized ommatidia with extra bristles (arrowheads in C5). (D) Histograms show mean percentage of flies eclosed from pupae of different genotypes: <i>ey-GAL4/+</i> (Black), <i>ey-GAL4/UAS-HA-imp-α3</i> (Grey), <i>ey-GAL4/+; UAS-Notch-ICD/+</i> (Green), and ey<i>-GAL4/UAS-HA-imp-α3; UAS-Notch-ICD/+</i> (Red). Note that 58% <i>ey-GAL4</i> driven Notch-ICD overexpressing pupae emerged as adult flies and this was reduced to 23% in which both <i>Notch-ICD</i> and <i>imp-α3</i> were overexpressed. The bars represent mean (± S.E.) of 9 replicates (n = 50 in each replicate; total n for each genotype = 450). (E) The survival curves of different genotypes: <i>ey-GAL4/+</i> (Black), <i>ey-GAL4/UAS-HA-imp-α3</i> (Grey), <i>ey-GAL4/+; UAS-Notch-ICD/+</i> (Green), and ey<i>-GAL4/UAS-HA-imp-α3; UAS-Notch-ICD/+</i> (Red). Note that both <i>HA-imp-α3</i> and <i>Notch-ICD</i> expressing flies showed significantly reduced life span as compared to only <i>HA-imp-α3</i> or <i>Notch-ICD</i> overexpressing flies.</p