The province is investing $1.5 million to support a housing project in Muskoka to help people with no place to call home.

An eight-year-old boy with good intentions sparked a missing person search on Tuesday.

After seven weeks, Honda Canada in Alliston will gradually begin operations next week.

Human remains located in Mulmur Township in March have been confirmed to be those of an 88-year-old Caledon man who was reported missing seven months ago.

The Ontario government gave garden centres and nurseries the green light to open their doors to the public on Friday.

Landlords who are missing thousands of dollars in rent or who find their properties damaged or strewn with garbage are concerned some tenants are taking advantage of the eviction ban put in place during the pandemic.

T.J. Lovell had just 30 minutes to pack up his belongings from the tent city in Oppenheimer Park if he wanted access to a hotel room that he could share with his father.

The following article, BREAKING: Sen Marsha Blackburn Introduces Stop COVID Act…Allowing US Citizens To Sue Communist China For Damage They’ve Inflicted On Our Nation, was first published on 100PercentFedUp.com.

Yesterday, Senator Marsha Blackburn (R-TN), along with Senator Martha McSally (R-AZ) introduced the Stop COVID Act, giving Americans the ability to sue Communist China for the damage they’ve inflicted on our nation. Senator Blackburn appeared on Fox News with host Judge Jeanine where she explained the act to Jeanine Pirro. Blackburn told the Fox News […]

Continue reading: BREAKING: Sen Marsha Blackburn Introduces Stop COVID Act…Allowing US Citizens To Sue Communist China For Damage They’ve Inflicted On Our Nation ...

The following article, Dem Congresswoman: Biden Allegation ‘Put To Rest’ Because He ‘Went on TV’ And Denied It, was first published on 100PercentFedUp.com.

Democrat Rep. Dina Titus says that the allegations against 2020 Democrat presidential candidate Joe Biden are “put to rest” because he “went on TV” and denied them. Rep. Titus spoke with MSNBC’s Ayman Mohyeldin today about how she believes Biden because he flatly said he didn’t do it and didn’t say he doesn’t remember. It’s […]

Continue reading: Dem Congresswoman: Biden Allegation ‘Put To Rest’ Because He ‘Went on TV’ And Denied It ...

The following article, ‘Schiff in Panic Mode’: Sources Say Russia Probe Transcripts Affirm Officials Found Nothing on Collusion, was first published on 100PercentFedUp.com.

Transcripts of House Intelligence Committee interviews have been cleared for release, and Adam Schiff is reportedly in panic mode. Unfortunately for Schiff, top law enforcement and intel officials said they saw no evidence of collusion between the Trump campaign and Russia in the transcripts. Republicans sought and got approval for the release of the transcripts […]

Continue reading: ‘Schiff in Panic Mode’: Sources Say Russia Probe Transcripts Affirm Officials Found Nothing on Collusion ...

The following article, Mother of the Year, Michelle Obama Explains How Having Kids Was A “Concession”… Had To Give Up Her “Aspirations and Dreams” [VIDEO], was first published on 100PercentFedUp.com.

While campaigning for her community organizer turned presidential candidate husband, Barack, Michelle Obama told a crowd of his supporters in Milwaukee, Wisconsin that for the first time in her life, she was proud to be an American. Four years later, Michelle Obama was a keynote speaker at the DNC convention, where she told Democrats how […]

Continue reading: Mother of the Year, Michelle Obama Explains How Having Kids Was A “Concession”… Had To Give Up Her “Aspirations and Dreams” [VIDEO] ...

The following article, Woman Shoots Three Teenage McDonald’s Workers For Telling Her She Couldn’t Eat In Dining Room Over COVID19 Restrictions, was first published on 100PercentFedUp.com.

An angry Oklahoma woman shot at a group of teenagers working at a local McDonald’s after they explained to her that the dining room was closed due to coronavirus restrictions. 32-year-old Gloricia Woody was arrested by the Oklahoma City Police and charged with the shooting. From the Oklahoma City Police -Last night, officers were called […]

Continue reading: Woman Shoots Three Teenage McDonald’s Workers For Telling Her She Couldn’t Eat In Dining Room Over COVID19 Restrictions ...

The following article, BREAKING: New Docs Prove Obama Knew Details Of Flynn Wiretapping…Newly Surfaced Video Shows Obama Explaining How He Stays Out Of FBI Investigations, was first published on 100PercentFedUp.com.

Barack Obama knew. Documents released yesterday that were used to exonerate President Trump’s new NSA General Flynn, prove that President Barack Obama was aware of the details of Michael Flynn’s intercepted phone calls on December 16 with then-Russian Ambassador Sergey Kislyak. On January 5, 2017, then-Deputy Attorney General, Sally Yates attended an Oval Office meeting […]

Continue reading: BREAKING: New Docs Prove Obama Knew Details Of Flynn Wiretapping…Newly Surfaced Video Shows Obama Explaining How He Stays Out Of FBI Investigations ...

The following article, BREAKING: President Trump’s Fiery Interview On Fox & Friends…”These are dirty politicians and dirty cops…They put our nation in danger with other nations, including Russia” [VIDEO], was first published on 100PercentFedUp.com.

This morning during a nearly one hour interview with Fox & Friends, President Trump addressed the decision by the DOJ to drop the case against the innocent General Michael Flynn. Trump ripped into the “dirty politicians and dirty cops” who went after General Michael Flynn. President Trump called the players involved in the horrible plot […]

Continue reading: BREAKING: President Trump’s Fiery Interview On Fox & Friends…”These are dirty politicians and dirty cops…They put our nation in danger with other nations, including Russia” [VIDEO] ...

In Lakeshore, it may be a little while longer before a retail cannabis store opens.

The provincial government has announced it will support child care centres that have been closed since March with their fixed operating costs as the fight against COVID-19 continues.

Little Richard, the self-proclaimed "architect of rock 'n' roll" whose piercing wail, pounding piano and towering pompadour irrevocably altered popular music while introducing black R&B to white America, has died Saturday. He was 87.

Research Event

Event participants

Speakers include:
Dr Lorand Bartels, Reader in International Law; Fellow, Trinity House, University of Cambridge
Laura Bannister, Senior Adviser on EU-UK Trade, Trade Justice Movement
Peter Holmes, Fellow, UKTPO; Reader in Economics, University of Sussex
Andrew Hood, Partner, Regulatory & Trade, FieldFisher LLP

Research Event

Event participants

Michael Gasiorek, Professor of Economics, University of Sussex; Director, Interanalysis; Fellow, UK Trade Policy Observatory, University of Sussex
Julia Magntorn Garrett, Research Officer, UK Trade Policy Observatory, University of Sussex
Prof Jim Rollo, Deputy Director, UK Trade Policy Observatory, University of Sussex; Associate Fellow, Global Economy and Finance Department, Chatham House
Nicolo Tamberi, Research Officer in the Economics of Brexit, University of Sussex
L. Alan Winters, Professor of Economics, Director, UK Trade Policy Observatory, University of Sussex

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue–absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical “second” cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS–based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent “bind-and-release” contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1–5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum. Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1.

The rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton–Valentine leukocidin (PVL) and α-toxin are exotoxins that create lytic pores in the host cell membrane. They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and α-toxin in their soluble, monomeric, and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C14PC). The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.

Tau is a microtubule-associated protein that plays a major role in Alzheimer's disease (AD) and other tauopathies. Recent reports indicate that, in the presence of crowding agents, tau can undergo liquid–liquid phase separation (LLPS), forming highly dynamic liquid droplets. Here, using recombinantly expressed proteins, turbidimetry, fluorescence microscopy imaging, and fluorescence recovery after photobleaching (FRAP) assays, we show that the divalent transition metal zinc strongly promotes this process, shifting the equilibrium phase boundary to lower protein or crowding agent concentrations. We observed no tau LLPS-promoting effect for any other divalent transition metal ions tested, including Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. We also demonstrate that multiple zinc-binding sites on tau are involved in the LLPS-promoting effect and provide insights into the mechanism of this process. Zinc concentration is highly elevated in AD brains, and this metal ion is believed to be an important player in the pathogenesis of this disease. Thus, the present findings bring a new dimension to understanding the relationship between zinc homeostasis and the pathogenic process in AD and related neurodegenerative disorders.

Mutations in retinaldehyde-binding protein 1 (RLBP1), encoding the visual cycle protein cellular retinaldehyde-binding protein (CRALBP), cause an autosomal recessive form of retinal degeneration. By binding to 11-cis-retinoid, CRALBP augments the isomerase activity of retinoid isomerohydrolase RPE65 (RPE65) and facilitates 11-cis-retinol oxidation to 11-cis-retinal. CRALBP also maintains the 11-cis configuration and protects against unwanted retinaldehyde activity. Studying a sibling pair that is compound heterozygous for mutations in RLBP1/CRALBP, here we expand the phenotype of affected individuals, elucidate a previously unreported phenotype in RLBP1/CRALBP carriers, and demonstrate consistencies between the affected individuals and Rlbp1/Cralbp−/− mice. In the RLBP1/CRALBP-affected individuals, nonrecordable rod-specific electroretinogram traces were recovered after prolonged dark adaptation. In ultrawide-field fundus images, we observed radially arranged puncta typical of RLBP1/CRALBP-associated disease. Spectral domain-optical coherence tomography (SD-OCT) revealed hyperreflective aberrations within photoreceptor-associated bands. In short-wavelength fundus autofluorescence (SW-AF) images, speckled hyperautofluorescence and mottling indicated macular involvement. In both the affected individuals and their asymptomatic carrier parents, reduced SW-AF intensities, measured as quantitative fundus autofluorescence (qAF), indicated chronic impairment in 11-cis-retinal availability and provided information on mutation severity. Hypertransmission of the SD-OCT signal into the choroid together with decreased near-infrared autofluorescence (NIR-AF) provided evidence for retinal pigment epithelial cell (RPE) involvement. In Rlbp1/Cralbp−/− mice, reduced 11-cis-retinal levels, qAF and NIR-AF intensities, and photoreceptor loss were consistent with the clinical presentation of the affected siblings. These findings indicate that RLBP1 mutations are associated with progressive disease involving RPE atrophy and photoreceptor cell degeneration. In asymptomatic carriers, qAF disclosed previously undetected visual cycle deficiency.

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Müller glia. In the RPE, isomerization of all-trans-retinyl esters to 11-cis-retinol is mediated by the retinoid isomerohydrolase Rpe65. A putative alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Müller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early, and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, while the emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaptation displayed significantly attenuated immediate photopic vision concomitant with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 min of light, early photopic vision was recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde supplementation. Genetic knockout of Des1 (degs1) or retinaldehyde-binding protein 1b (rlbp1b) did not eliminate photopic vision in zebrafish. Our findings define molecular and temporal requirements of the nonphotopic or photopic visual cycles for mediating vision in bright light.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation. We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo. Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42 ± 19% reduction in tissue respiration rate and a 33 ± 38% reduction in CcO activity compared with unexposed mice. Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC–treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 μm) and nitric oxide (100 μm). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 μm, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC or other CO scavengers demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

20 November 2019

5 December 2019

Invitation Only Research Event

Event participants

Ali Farhan Hamid, Governor of Anbar Province
Chair: Dr Renad Mansour, Senior Research Fellow, Middle East and North Africa Programme, Chatham House

6 January 2020

Invitation Only Research Event

Event participants

Chair: Dr Lina Khatib, Director, Middle East and North Africa Programme, Chatham House

21 January 2020

Discussions of North African integration have evoked ideas of a shared identity and a common destiny in the region. However, recent attempts to build regional blocs in North Africa have been unsuccessful. This paper examines the benefits of a ‘synergistic’ approach to North African cooperation. 

Invitation Only Research Event

Event participants

Usama Otman Essed, Libya Center for Strategic & Future Studies
Chair: Tim Eaton, Middle East and North Africa Programme, Chatham House

Invitation Only Research Event

26 February 2020

26 February 2020

Over the past two decades, China has increased its presence in North Africa in terms of trade and investment. This paper looks at China’s policy within the context of its Africa and Middle East policies to better understand its approach to Morocco and Tunisia.

Research Event

Event participants

Research Event

Event participants

Glada Lahn, Senior Research Fellow, Energy, Environment and Resources Programme, Chatham House
Greg Shapland, Associate Fellow, Middle East and North Africa Programme, Chatham House 
Moderator: Sanam Vakil, Deputy Director and Senior Research Fellow, Middle East and North Africa Programme, Chatham House