The effect of tertiary treated wastewater on fish growth and health: Laboratory-scale experiment with Poecilia reticulata (guppy)

Treated wastewater (TWW) constitutes a sustainable water resource and has been used for fish culture in some countries around the world, although there are no comprehensive data on the effect of TWW on fish growth and health in the context of aquaculture production. Our objectives were to examine how fish culture in TWW affected fish growth and fitness, as well as compliance with the international standards for safe consumption. Guppy (Poecilia reticulata) fingerlings were reared in 0%, 50% and 100% tertiary TWW (TTWW), from the age of five days, for a period of four months. In water analyses, 33 out of 67 tested organic micropollutants (OMPs) were detected in the TTWW samples at least once, at concentrations that are typically reported in domestic TTWW. Fish survival ranged between 77-80% and did not differ between treatment groups. Fish growth and mortality following challenge infection with Tetrahymena sp. (which ranged between 64-68%), were similar among treatment groups. Of tested immunological parameters, lysozyme and anti-protease was similar among treatments while complement activity was highest in the 50% TTWW-reared fish. No abnormalities were observed in the histopathological analysis. Levels of heavy metals, poly-chlorinated-biphenyls (PCBs) and organochlorines (OCs) in fish were below the detection limit and below the Food and Agriculture Organization of the United Nations (FAO) and the European Union EU maximal permitted levels in food fish. Results suggest that the yield of fish grown in TTWW is potentially similar to that in freshwater, and the produced fish comply with the standards of consumer safety. The results are in line with previous studies that examined the feasibility of TWW-fed aquaculture.Jewish Charitable Association (ICA); Israel Ministry of Science and Technology [3-12876]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Preclinical Safety Evaluation in Rats of a Polymeric Matrix Containing an siRNA Drug Used as a Local and Prolonged Delivery System for Pancreatic Cancer Therapy

Conventional chemotherapy treatments for pancreatic cancer are mainly palliative. RNA interference (RNAi)-based drugs present the potential for a new targeted treatment. LOcal Drug EluteR (LODERTM) is a novel biodegradable polymeric matrix that shields drugs against enzymatic degradation and releases small interfering RNA (siRNA) against G12D-mutated KRAS (siG12D). siG12D-LODER has successfully passed a phase 1/2a clinical trial. Such a formulation necessitates biocompatibility and safety studies. We describe the safety and toxicity studies with siG12D-LODER in 192 Hsd:Sprague Dawley rats, after repeated subcutaneous administrations (days 1, 14, and 28). Animals were sacrificed on days 29 and 42 (recovery phase). In all groups, no adverse effects were noted, and all animals showed favorable local and systemic tolerability. Histopathologically, LODER implantation resulted in the expected capsule formation, composed of a thin fibrotic tissue. On the interface between the cavity and the capsule, a single layer composed of macrophages and multinucleated giant cells was observed. No difference was noted between the placebo and siG12D-LODER groups. These findings provide valuable information for future preclinical studies with siRNA-bearing biodegradable polymers and for the safety aspects of RNAi-based drugs as a targeted therapy

Membrane anchored IL-18 linked to constitutively active TLR4 and CD40 improves human T cell antitumor capacities for adoptive cell therapy

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TILs) or blood T cells genetically redirected by an antitumor TCR or CAR induces a strong antitumor response in a proportion of patients with cancer; however, the therapeutic efficacy is often limited by rapid decline in T cell functions. Coadministering supportive cytokines frequently provokes systemic side effects preventing their broad clinical application. We recently showed that cytokines can be anchored to the cell membrane in a functional fashion and that cytokine receptor signaling can synergize with TLR4 and CD40 signaling. Here, we aimed at augmenting T cell activation by simultaneous signaling through the cytokine receptor, toll-like receptor and TNF-type receptor using IL-18, TLR4 and CD40 as prototypes. METHODS: Genes were expressed on electroporation of in vitro-transcribed mRNA in CD4(+) and CD8(+) T cells from healthy donors redirected against melanoma cells with an anti-melanotransferrin CAR and in TILs derived from melanoma patients. Functional assays included the activation of signaling pathways, expression of activation and differentiation markers, cytokine secretion and killing of melanoma target cells. RESULTS: To provide IL-18 costimulation to T cells in-cis while avoiding systemic effects, we genetically anchored IL-18 to the T cell membrane, either alone (memIL-18) or fused with constitutively active (ca)TLR4 and caCD40 signaling domains arranged in tandem, creating a synthetic ‘all-in-one’ memIL-18-TLR4-CD40 receptor. MemIL-18-TLR4-CD40, but not memIL-18, triggered strong NF-κB activation in cells lacking the IL-18 receptor, attesting to functionality of the TLR-CD40 moiety. While the membrane-anchored cytokine was found to act mainly in-cis, some T cell activation in-trans was also observed. The electroporated T cells exhibited spontaneous T-bet upregulation and IFN-γ and TNF-α secretion. Melanoma-induced activation of CAR-T cells and TILs as manifested by cytokine secretion and cytolytic activity was substantially augmented by both constructs, with memIL-18-TLR4-CD40 exerting stronger effects than memIL-18 alone. CONCLUSIONS: Linking membrane anchored IL-18 with caTLR4 and caCD40 signaling in one hybrid transmembrane protein provides simultaneous activation of three T cell costimulatory pathways through one genetically engineered membrane molecule, strongly amplifying T cell functions for adoptive T cell therapy of cancer

MYC is a clinically significant driver of mTOR inhibitor resistance in breast cancer

Targeting the PI3K–AKT–mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K–AKT–mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies

Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness

Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity

food and memory

ניסוי במסגרת הקורס בפסיכולוגיה ניסויית קיץ 201

CLKGR is present in the nucleus, and competitively inhibits CLK function.

<p><b>A.</b> CLKGR protein is expressed in large amounts in TIM-CLKGR flies. Western blot from fly heads collected at CT15 using an anti-CLK antibody. The assay was performed from TIM-CLKGR flies and control flies (<i>tim-gal4/+</i>). Arrows indicate CLK or the CLKGR fusion protein, which can be distinguished by their size. <b>B.</b> CLKGR is present in both the nuclei and cytoplasm in TIM-CLKGR flies. Western blot from nuclear and cytoplasmic extracts of control (<i>tim-gal4/+</i>) or TIM-CLKGR fly heads collected at CT15. TUBULIN staining is shown as negative control for the nuclear fraction separation and positive control for the cytoplasm fraction. HISTONE-3 staining is shown as positive control for nuclear separation and negative control for the cytoplasm separation. <b>C.</b> CLKGR expression can inhibit CLK-mediated activity in <i>Drosophila</i> S2 cells. <i>Drosophila</i> S2 cells were transfected with <i>vri-luciferase</i> reporter plasmid, pAc-CLK plasmid, a plasmid that express CLK or CLKGR under regulation of a copper inducible promoter (metallothionein; MT-CLK or MT-CLKGR respectively), and a plasmid used for controlling transfection efficiency (pCopia-Renilla). No copper or two different amounts of copper were utilized as indicated in the graph. Experiment was done at three separate biological repeats. Plot shows average values of biological duplicates of one representative experimental repeat. Error bars represent standard deviation. One-way Anova was performed to determine statistical significance. *<i>p</i><0.05. <b>D.</b> CLKGR can bind to CLK targets promoters. We induced CLKGR expression in S2 cells using the MT-CLKGR plasmid; in parallel to constant expression of CYCVP16 (from the pAc-CYCVP16 expressing plasmid). CYCVP16 and CLKGR together activate CLK-driven transcription suggesting they bind to CLK targets promoters. Experiment was done at three separate biological repeats. Plot shows average values of duplicates of one representing repeat. Error bars indicate standard deviation. CLK-target activity was measured using a <i>vri</i>-luciferase reporter and values were normalized to a transfection control (pCopia-Renilla). T-test was performed to determine statistical significance between time points. ***<i>p</i><0.001.</p

Synergistic Interactions between the Molecular and Neuronal Circadian Networks Drive Robust Behavioral Circadian Rhythms in <i>Drosophila melanogaster</i>

<div><p>Most organisms use 24-hr circadian clocks to keep temporal order and anticipate daily environmental changes. In <i>Drosophila melanogaster</i> CLOCK (CLK) and CYCLE (CYC) initiates the circadian system by promoting rhythmic transcription of hundreds of genes. However, it is still not clear whether high amplitude transcriptional oscillations are essential for circadian timekeeping. In order to address this issue, we generated flies in which the amplitude of CLK-driven transcription can be reduced partially (approx. 60%) or strongly (90%) without affecting the average levels of CLK-target genes. The impaired transcriptional oscillations lead to low amplitude protein oscillations that were not sufficient to drive outputs of peripheral oscillators. However, circadian rhythms in locomotor activity were resistant to partial reduction in transcriptional and protein oscillations. We found that the resilience of the brain oscillator is depending on the neuronal communication among circadian neurons in the brain. Indeed, the capacity of the brain oscillator to overcome low amplitude transcriptional oscillations depends on the action of the neuropeptide PDF and on the <i>pdf</i>-expressing cells having equal or higher amplitude of molecular rhythms than the rest of the circadian neuronal groups in the fly brain. Therefore, our work reveals the importance of high amplitude transcriptional oscillations for cell-autonomous circadian timekeeping. Moreover, we demonstrate that the circadian neuronal network is an essential buffering system that protects against changes in circadian transcription in the brain.</p></div

Behavioral characterization of flies expressing CLKGR.

<p>Behavioral analysis of flies maintained for 10 days in constant darkness (DD 1–10). Fly strains: TIM-CLKGR (<i>tim-gal4</i>; UAS-<i>ClkGR</i>), TIM-CLKGR(X2) (<i>tim-gal4</i>; UAS-<i>ClkGR/</i>UAS-<i>ClkGR</i>), TIMGAL4 (<i>tim-gal4/+</i>) and UAS-CLKGR (UAS-<i>ClkGR/+</i>). Period of rhythmic flies, rhythmic flies percentage and average power were calculated by chi square power p<0.05. SEM is shown in brackets.</p