G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of ...processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.
Astrocytes become reactive in response to insults to the central nervous system by adopting context-specific cellular signatures and outputs, but a systematic understanding of the underlying ...molecular mechanisms is lacking. In this study, we developed CRISPR interference screening in human induced pluripotent stem cell-derived astrocytes coupled to single-cell transcriptomics to systematically interrogate cytokine-induced inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures, one promoted by STAT3 and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in human brains in Alzheimer's disease and hypoxic-ischemic encephalopathy. Furthermore, we validated that markers of these signatures were regulated by STAT3 in vivo using a mouse model of neuroinflammation. These results and the platform that we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.
Objective
This study was performed to evaluate the therapeutic effect and diagnostic value of a novel nasopharyngeal stent (Naśtent; Corinium Medical Equipment Ltd., Cirencester, UK). The Naśtent is ...designed to stent palatal collapse in patients with sleep-related breathing disorders.
Methods
The study was conducted from 2018 to 2019. Patients who did not qualify for continuous positive airway pressure therapy underwent split-night examination with an inserted Naśtent for the first half of the night. The next morning, drug-induced sleep endoscopy (DISE) was performed.
Results
Of the 122 enrolled patients, 21 were excluded because of Naśtent intolerance (n = 14) or technically invalid examinations (n = 7). Among the remaining 101 patients, in correlation with DISE, the apnea–hypopnea index was significantly reduced in patients with palatal obstructions, mainly in those with anteroposterior collapse patterns. The Naśtent did not influence retrolingual or multilevel obstructions. Using a 40% reduction of the apnea–hypopnea index by the Naśtent as a cut-off value, 85.7% of soft palate obstructions were detected compared with DISE.
Conclusions
The Naśtent is a viable tool to reduce palatal obstructions, although it is not readily tolerated. It may also be helpful for diagnosis of sleep-related breathing disorders when DISE is unavailable.
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in ...microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
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► Ultracomplex shRNA library minimizes false positives/negatives in genome-wide screens ► Pooled double-shRNA strategy systematically maps genetic interactions between hits ► Application of two-step strategy identifies pathways controlling ricin susceptibility ► The resulting map uncovers functionally distinct mammalian TRAPP complexes
A high-throughput method that relies on the use of ultracomplex shRNA libraries makes it possible to create genetic interaction maps in mammalian cells. This approach will be applicable to many cellular processes and conditions, as illustrated by the discovery of distinct TRAPP complexes involved in endocytosis.
Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function. Pathological transformation of astrocytes to reactive states can be protective or harmful ...to BBB function. Here, using a human induced pluripotent stem cell (iPSC)-derived BBB co-culture model, we show that tumor necrosis factor (TNF) transitions astrocytes to an inflammatory reactive state that causes BBB dysfunction through activation of STAT3 and increased expression of SERPINA3, which encodes alpha 1-antichymotrypsin (α1ACT). To contextualize these findings, we correlated astrocytic STAT3 activation to vascular inflammation in postmortem human tissue. Further, in murine brain organotypic cultures, astrocyte-specific silencing of Serpina3n reduced vascular inflammation after TNF challenge. Last, treatment with recombinant Serpina3n in both ex vivo explant cultures and in vivo was sufficient to induce BBB dysfunction-related molecular changes. Overall, our results define the TNF-STAT3-α1ACT signaling axis as a driver of an inflammatory reactive astrocyte signature that contributes to BBB dysfunction.
Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously ...identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles.
The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting ...nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.
Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical ...pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.
Hsp70 is a stress-inducible molecular chaperone that is required for cancer development at several steps. Targeting the active site of Hsp70 has proven relatively challenging, driving interest in ...alternative approaches. Hsp70 collaborates with the Bcl2-associated athanogene 3 (Bag3) to promote cell survival through multiple pathways, including FoxM1. Therefore, inhibitors of the Hsp70-Bag3 protein-protein interaction (PPI) may provide a noncanonical way to target this chaperone. We report that JG-98, an allosteric inhibitor of this PPI, indeed has antiproliferative activity (EC50 values between 0.3 and 4 μmol/L) across cancer cell lines from multiple origins. JG-98 destabilized FoxM1 and relieved suppression of downstream effectors, including p21 and p27. On the basis of these findings, JG-98 was evaluated in mice for pharmacokinetics, tolerability, and activity in two xenograft models. The results suggested that the Hsp70-Bag3 interaction may be a promising, new target for anticancer therapy.
A pilot plant for ozonation and UV-disinfection received effluent from a German municipal sewage treatment plant (STP) to test the removal of pharmaceuticals, iodinated X-ray contrast media (ICM) and ...musk fragrances from municipal wastewater. In the original STP effluent, 5 antibiotics (0.34–0.63
μg
l
−1), 5 betablockers (0.18–1.7
μg
l
−1), 4 antiphlogistics (0.10–1.3
μg
l
−1), 2 lipid regulator metabolites (0.12–0.13
μg
l
−1), the antiepileptic drug carbamazepine (2.1
μg
l
−1), 4 ICM (1.1–5.2
μg
l
−1), the natural estrogen estrone (0.015
μg
l
−1) and 2 musk fragrances (0.1–0.73
μg
l
−1) were detected by LC-electrospray tandem MS and/or GC/MS/MS. ICM, derived from radiological examinations, were present with the highest concentrations (diatrizoate: 5.7
μg
l
−1, iopromide: 5.2
μg
l
−1). By applying 10–15
mg
l
−1 ozone (contact time: 18
min), all the pharmaceuticals investigated as well as musk fragrances (HHCB, AHTN) and estrone were no longer detected. However, ICM (diatrizoate, iopamidol, iopromide and iomeprol) were still detected in appreciable concentrations. Even with a 15
mg
l
−1 ozone dose, the ionic diatrizoate only exhibited removal efficiencies of not higher than 14%, while the non-ionic ICM were removed to a degree of higher than 80%. Advanced oxidation processes (O
3/UV-low pressure mercury arc, O
3/H
2O
2), which were non-optimized for wastewater treatment, did not lead significantly to a higher removal efficiency for the ICM than ozone alone.
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