Rhenium fac-Tricarbonyl Bisimine Chalcogenide Complexes: Synthesis, Photophysical Studies, and Confocal and Time-Resolved Cell Microscopy.

We describe the preparation, characterization, and imaging studies of rhenium carbonyl complexes with a pyta (4-(2-pyridyl)-1,2,3-triazole) or tapy (1-(2-pyridyl)-1,2,3-triazole)-based heteroaromatic N∧N ligand and thiolate or selenoate X ligand. The stability and photophysical properties of the selenolate complexes are compared with parent chloride complexes and previously described analogues with benzenethiolate ligands. Two complexes were imaged in A549 cells upon excitation at 405 nm. Colocalization studies suggest a lysosomal accumulation, while one parent chloride complex was described to localize at the Golgi apparatus. Preliminary fluorescence lifetime measurements and imaging demonstrate potential for application in time-resolved microscopy techniques due to the long and variable lifetimes observed in cellular environments, including an increase in lifetime between the solution and solid state many times larger than previously reported.

A New Manganese Superoxide Dismutase Mimetic Improves Oxaliplatin-Induced Neuropathy and Global Tolerance in Mice.

Reactive oxygen species (ROS) are produced by every aerobic cell during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Superoxide Dismutases (SOD) are antioxidant proteins that convert superoxide anions (O2•-) to hydrogen peroxide (H2O2) and dioxygen. Using the differential in the level of oxidative stress between normal and cancer cells, SOD mimetics can show an antitumoral effect and prevent oxaliplatin-induced peripheral neuropathy. New Pt(IV) conjugate prodrugs (OxPt-x-Mn1C1A (x = 1, 1-OH, 2)), combining oxaliplatin and a Mn SOD mimic (MnSODm Mn1C1A) with a covalent link, were designed. Their stability in buffer and in the presence of sodium ascorbate was studied. In vitro, their antitumoral activity was assessed by the viability and ROS production of tumor cell lines (CT16, HCT 116, KC) and fibroblasts (primary culture and NIH 3T3). In vivo, a murine model of colorectal cancer was created with subcutaneous injection of CT26 cells in Balb/c mice. Tumor size and volume were measured weekly in four groups: vehicle, oxaliplatin, and oxaliplatin associated with MnSODm Mn1C1A and the bis-conjugate OxPt-2-Mn1C1A. Oxaliplatin-induced peripheral neuropathy (OIPN) was assessed using a Von Frey test reflecting chronic hypoalgesia. Tolerance to treatment was assessed with a clinical score including four items: weight loss, weariness, alopecia, and diarrhea. In vitro, Mn1C1A associated with oxaliplatin and Pt(IV) conjugates treatment induced significantly higher production of H2O2 in all cell lines and showed a significant improvement of the antitumoral efficacy compared to oxaliplatin alone. In vivo, the association of Mn1C1A to oxaliplatin did not decrease its antitumoral activity, while OxPt-2-Mn1C1A had lower antitumoral activity than oxaliplatin alone. Mn1C1A associated with oxaliplatin significantly decreased OIPN and also improved global clinical tolerance of oxaliplatin. A neuroprotective effect was observed, associated with a significantly improved tolerance to oxaliplatin without impairing its antitumoral activity.

Chronic Low Back Pain Forced Me to Search for and Find Pain Solutions: An Autobiographical Case Report.

Lifelong, pregnancy-induced low back pain forced me to search for solutions to the problem of pain. Currently, low back pain is often diagnosed as "nonspecific" and, as a result, a multitude of tests and poorly effective, at times side effect-laden or habit-forming treatments are recommended. My quest for relief took me to first diagnose my pain as coming from the sacroiliac joints, then to prolotherapy, the first treatment which brought me prolonged relief. I then learned how to perform prolotherapy. In 2009, when I undertook a randomized controlled study of dextrose prolotherapy for rotator cuff tendinopathy, I restricted my practice to treating pain. As low back pain was a large part of my practice, I sought new ways to examine the sacroiliac joints. I conducted a consecutive patient data collection which suggested that over three-quarters of those with low back pain suffer from displaced sacroiliac joints. In a further randomized controlled study, I found that the two-minute corrective exercise I derived from this test provided immediate relief to 90% of those using it. With Dr. John Clark Lyftogt I discovered the safety and effectiveness of 5% dextrose perineural injections to provide immediate pain relief to any area supplied by a nerve I could reach with my needle. As I was treating many diabetics with peripheral neuropathy, I shifted my perineural injection material to 5% mannitol, which may be as effective, with less exposure to dextrose as a potential benefit for diabetics. As most people dislike injections, a pharmacist and I developed a mannitol-containing topical cream for pain relief. We compared a base cream to the same cream with mannitol on lips pretreated with capsaicin cream which made them burn. By 10 minutes the probability the two creams were as effective in relieving the burn was less than 0.001 in favor of mannitol. When given to 235 patients with a total of 289 different painful conditions, we found that it provided 53% relief in an average of 16 minutes with a median of four hours duration. Now retired, after 55 years of medical practice, I love to relieve the pain of friends and fellow hikers using exercise and cream. Searching for and finding solutions to chronic pain has enriched my life and that of many others.

SOD mimics: From the tool box of the chemists to cellular studies.

Superoxide dismutases (SODs) are metalloproteins that protect cells against oxidative stress by controlling the concentration of superoxide (O2-) through catalysis of its dismutation. The activity of superoxide dismutases can be mimicked by low-molecular-weight complexes having potential therapeutic applications. This review presents recent strategies for designing efficient SOD mimics, from molecular metal complexes to nanomaterials. Studies of these systems in cells reveal that some SOD mimics, designed to react directly with superoxide, may also indirectly enhance the cellular antioxidant arsenal. Finally, a good understanding of the bioactivity requires information on the cell-penetration, speciation, and subcellular location of the SOD mimics: we will describe recent studies and new techniques that open opportunities for characterizing SOD mimics in biological environments.

Cellular Detection of a Mitochondria Targeted Brominated Vinyl Triphenylamine Optical Probe (TP-Br) by X-Ray Fluorescence Microscopy.

Triphenylamine (TP) derivatives such as two-branch cationic vinylbenzimidazolium triphenylamine TP-2Bzim are promising turn-on fluorescent probes suitable for two-photon imaging, labelling mitochondria in live cells. Here, we designed two TP-2Bzim derivatives as bimodal probes suitable for X-ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP-RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP-Br probe retaining good photophysical properties and mitochondria labelling in live cells. The influence of calcium channels in the uptake of TP-Br was studied. Synchrotron Radiation X-ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP-Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.

Self-Treatment of Chronic Low Back Pain Based on a Rapid and Objective Sacroiliac Asymmetry Test: A Pilot Study.

BACKGROUND: Low back pain (LBP) is common, costly, and disabling. This study assesses a novel and simple LBP evaluation method and its merit in guiding the direction of a self-treatment exercise. METHODS: Randomized open-label intention is used to treat the study. Consecutive patients with LBP ≥ three months and pain ≥ 5/10 were evaluated in a Vancouver clinic with the sacroiliac forward flexion test (SIFFT) by comparing the height of posterior superior iliac spines using a level. Those with asymmetry ≥ 5 mm were offered participation. The assistant, who generated and encrypted the randomization, assigned participants: group 1 learned a two-minute, SIFFT-derived, sacroiliac-leveling exercise (SIFFT-E) as needed for LBP relief; group 2 used a pelvic stabilization belt as needed to prevent LBP, and group 3 continued the usual care. After one month, all participants used SIFFT-E and belt as needed for one month. The identifier number of this article in Clinicaltrials.gov is #NCT03888235. The trial is closed. Our primary outcome measure was the Oswestry disability index (ODI) (decrease) from baseline to one and two months. We also followed SIFFT improvement (decrease). FINDINGS: Of 72 LBP patients, 62 (86%) had ≥ 5 mm asymmetry. From zero to one month, the 21 (one dropout) SIFFT-E participants outperformed the 20 usual care participants for ODI improvement (12.5 ± 14.8 vs. -3.4 ± 14.9 points; mean difference 15.9 [CI 6.7-25.0]; P = 0.002 with number needed to treat (NNT) of 3.0 for ODI improvement ≥ 11). Belt use results were intermediate. At two months, after all the 62 participants used the exercise and belt as needed, combined ODI improvements were clinically significant (12.0 ± 18.4 points), and SIFFT asymmetry was reduced by 8.6 ± 8.6 mm. Five (8%) exercise and 12 (19%) belt wearers experienced mild side effects. INTERPRETATION: Sacroiliac asymmetry appears to be frequent. SIFFT may be clinically useful as an evaluation tool for prescribing a simple self-directed corrective exercise as seen by clinically significant improvements in function and asymmetry.

Clinical Variability of SYNJ1-Associated Early-Onset Parkinsonism.

Autosomal recessive early-onset parkinsonism is clinically and genetically heterogeneous. Mutations of three genes, PRKN, PINK1, and DJ-1 cause pure phenotypes usually characterized by levodopa-responsive Parkinson's disease. By contrast, mutations of other genes, including ATP13A2, PLA2G6, FBXO7, DNAJC6, SYNJ1, VPS13C, and PTRHD1, cause rarer, more severe diseases with a poor response to levodopa, generally with additional atypical features. We performed data mining on a gene panel or whole-exome sequencing in 460 index cases with early-onset (≤ 40 years) Parkinson's disease, including 57 with autosomal recessive disease and 403 isolated cases. We identified two isolated cases carrying biallelic mutations of SYNJ1 (double-heterozygous p.D791fs/p.Y232H and homozygous p. Y832C mutations) and two siblings with the recurrent homozygous p.R258Q mutation. All four variants were absent or rare in the Genome Aggregation Database, were predicted to be deleterious on in silico analysis and were found to be highly conserved between species. The patient with both the previously unknown p.D791fs and p.Y232H mutations presented with dystonia-parkinsonism accompanied by a frontal syndrome and oculomotor disturbances at the age of 39. In addition, two siblings from an Algerian consanguineous family carried the homozygous p.R258Q mutation and presented generalized tonic-clonic seizures during childhood, with severe intellectual disability, followed by progressive parkinsonism during their teens. By contrast, the isolated patient with the homozygous p. Y832C mutation, diagnosed at the age of 20, had typical parkinsonism, with no atypical symptoms and slow disease progression. Our findings expand the mutational spectrum and phenotypic profile of SYNJ1-related parkinsonism.

Gene Panel Sequencing Identifies Novel Pathogenic Mutations in Moroccan Patients with Familial Parkinson Disease.

In the past two decades, genetic studies of familial forms of Parkinson's disease (PD) have shown evidence that PD has a significant genetic component. Indeed, 12 genes are strongly involved in PD causality, three of them having dominant inheritance and 9 causing early-onset autosomal recessive forms, including 3 with a typical PD and 6 with an atypical parkinsonism. The aim of this study was to determine the genetic basis of familial PD in Moroccan patients. We selected 18 Moroccan index case with familial forms of PD. Patients were first screened for exon-rearrangements by MLPA kit. They were then analyzed by gene panel next-generation sequencing (NGS). Functional variants with minor allele frequencies < 0.5% in public databases were considered potential candidate variants to PD. In the 18 PD patients with a positive family history that were analyzed, MLPA assays identified PRKN deletions in two patients: a homozygous exon 3-5 deletion and a heterozygous exon 4 deletion. Sixteen rare SNV were identified by NGS, four of them were novel. Seven mutations were categorized as pathogenic, five as likely pathogenic, two to be of uncertain significance, and 3 were predicted to be likely benign but may give a weaker pathogenic effect and could contribute to PD since they were found in late-onset PD patients. Rare or novel mutations that could be related to the disease were identified in 72% of these patients (13/18), including nine with bi-allelic pathogenic/likely pathogenic variants in genes causing recessive PD, particularly PRKN and PINK1. Mutations in genes with dominant inheritance were found in 4/18 patients (22%).

Understanding the g-tensors of perchlorotriphenylmethyl and Finland-type trityl radicals.

The 285 GHz EPR spectra of perchlorotriphenylmethyl and tetrathiatriarylmethyl radicals in frozen solution have been accurately measured. The relationship between their molecular structures and their g-tensors has been investigated with the aid of DFT calculations, revealing that the degree of spin density delocalization away from the central methylene carbon is an important determining factor of the g-anisotropy. In particular, the small amount of spin densities on the Cl or S heteroatoms at the 2 and 6 positions with respect to the central carbon have the strongest influence. Furthermore, the amount of spin densities on these heteroatoms and thus the anisotropy can be modulated by the protonation (esterification) state of the carboxylate groups at the 4 position. These results provide unique insights into the g-anisotropy of persistent trityl radicals and how it can be tuned.

Genetic and Phenotypic Basis of Autosomal Dominant Parkinson's Disease in a Large Multi-Center Cohort.

LRRK2, SNCA, and VPS35 are unequivocally associated with autosomal dominant Parkinson's disease (PD). We evaluated the prevalence of LRRK2, SNCA, and VPS35 mutations and associated clinical features in a large French multi-center cohort of PD patients. Demographic and clinical data were collected for 1,805 index cases (592 with autosomal dominant inheritance and 1,213 isolated cases) since 1990. All probands were screened with TaqMan assays for LRRK2 Gly2019Ser. In the absence of this mutation, the coding sequences of the three genes were analyzed by Sanger sequencing and/or next-generation sequencing. The data for the three genes were analyzed according to age at onset, family history, ethnic origin and clinical features. We identified 160 index cases (8.9%) with known pathogenic variants: 138 with pathogenic LRRK2 variants (7.6%), including 136 with the Gly2019Ser mutation, 19 with SNCA point mutations or genomic rearrangements (1.1%), and three with the VPS35 Asp620Asn mutation (0.16%). Mutation frequencies were higher in familial than isolated cases, consistent with autosomal dominant inheritance (12.0 vs. 7.3%; OR 1.7, 95% CI [1.2-2.4], p = 0.001). PD patients with LRRK2 variants were more likely to have higher rates of late-onset PD (>50 years; OR 1.5, 95% CI [1.0-2.1], p = 0.03), whereas those with SNCA mutations tended to have earlier age at onset disease (≤ 50 years, p = 0.06). The clinical features of LRRK2 carriers and those without any pathogenic variants in known PD-associated genes were similar. The likelihood of detecting disease-causing mutations was higher in cases compatible with autosomal dominant inheritance.

Exome Sequencing Reveals Signal Transduction Genes Involved in Impulse Control Disorders in Parkinson's Disease.

Introduction: Impulse control disorders (ICDs) frequently complicate dopamine agonist (DA) therapy in Parkinson's disease (PD). There is growing evidence of a high heritability for ICDs in the general population and in PD. Variants on genes belonging to the reward pathway have been shown to account for part of this heritability. We aimed to identify new pathways associated with ICDs in PD. Methods: Thirty-six Parkinsonian patients on DA therapy with (n = 18) and without ICDs (n = 18) matched on age at PD's onset, and gender was selected to represent the most extreme phenotypes of their category. Exome sequencing was performed, and variants with a strong functional impact in brain-expressed genes were selected. Allele frequencies and their distribution in genes and pathways were analyzed with single variant and SKAT-O tests. The 10 most associated variants, genes, and pathways were retained for replication in the Parkinson's progression markers initiative (PPMI) cohort. Results: None of markers tested passed the significance threshold adjusted for multiple comparisons. However, the "Adenylate cyclase activating" pathway, one of the top associated pathways in the discovery data set (p = 1.6 × 10-3) was replicated in the PPMI cohort and was significantly associated with ICDs in a post hoc pooled analysis (combined p-value 3.3 × 10-5). Two of the 10 most associated variants belonged to genes implicated in cAMP and ERK signaling (rs34193571 in RasGRF2, p = 5 × 10-4; rs1877652 in PDE2A, p = 8 × 10-4) although non-significant after Bonferroni correction. Conclusion: Our results suggest that genes implicated in the signaling pathways linked to G protein-coupled receptors participate to genetic susceptibility to ICDs in PD.

Anti-inflammatory activity of superoxide dismutase mimics functionalized with cell-penetrating peptides.

A superoxide dismutase mimic (Mn1) was functionalized with three positively charged-peptides: RRRRRRRRR (Mn1-R9), RRWWWRRWRR (Mn1-RW9) or Fx-r-Fx-K (Mn1-MPP). Characterization of the physico-chemical properties of the complexes show that they share similar binding affinity for Mn2+, apparent reduction potential and intrinsic superoxide dismutase activity. However, their accumulation in cells is different (Mn1-R9 < Mn1-MPP < Mn1-RW9 < Mn1), as well as their subcellular distribution. In addition, the three functionalized-complexes display a better anti-inflammatory activity than Mn1 when assayed at 10 µM. This improvement is due to a combination of an anti-inflammatory effect of the peptidyl moiety itself, and of the SOD mimic for Mn1-RW9 and Mn1-MPP. In contrast, the enhanced anti-inflammatory activity of Mn1-R9 is solely due to the SOD mimic.

Oxaliplatin-induced neuropathy: the preventive effect of a new super-oxide dismutase modulator.

By using the differential in level of oxidative status between normal and cancer cells, SuperOxide Dismutase (SOD) mimetics can have anti-tumor efficacy and prevent oxaliplatin-induced peripheral neuropathy. Our objective was to evaluate the neuroprotective efficacy of MAG, a new SOD mimic. In vitro, the effects of MAG alone or with oxaliplatin were studied on colon cancer cells (HT29 and CT26) and on normal fibroblast cells (NIH3T3). The cell viability (by crystal violet) as well as the production of reactive forms of oxygen and glutathione (by spectrofluorimetric assay) was measured. In vivo, efficacy on tumor growth was assessed in mice grafted with CT26 colon cancer cells. The effects on induced neurotoxicity were measured by specific behavioral Von Frey nociception, cold-plate tests, specific functional neuromuscular assay and electron microscopy. In vitro, MAG induced a production of hydrogen peroxide in all cells. At 24 h-incubation, MAG exhibits a cytotoxic activity in all cell lines. A cytotoxic additive effect of MAG and oxaliplatin was observed through oxidative burst. In vivo, oxaliplatin-treated mice associated with MAG did not counteract oxaliplatin's antitumoral efficacy. After 4 weeks of treatment with oxaliplatin combined with MAG, behavioral and functional tests showed a decrease in peripheral neuropathy induced by oxaliplatin in vivo. Electron microscopy analyses on sciatic nerves revealed an oxaliplatin-induced demyelination which is prevented by the association of MAG to this chemotherapy. In conclusion, MAG prevents the appearance of sensitive axonal neuropathy and neuromuscular disorders induced by oxaliplatin without affecting its antitumor activity.

From Naproxen Repurposing to Naproxen Analogues and Their Antiviral Activity against Influenza A Virus.

The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.

Labeling of Hyaluronic Acids with a Rhenium-tricarbonyl Tag and Percutaneous Penetration Studied by Multimodal Imaging.

Hyaluronic acids were labeled with a rhenium-tricarbonyl used as single core multimodal probe for imaging and their penetration into human skin biopsies was studied using IR microscopy and fluorescence imaging (labeled SCoMPI). The penetration was shown to be dependent on the molecular weight of the molecule and limited to the upper layer of the skin.

Reversible Keap1 inhibitors are preferential pharmacological tools to modulate cellular mitophagy.

Mitophagy orchestrates the autophagic degradation of dysfunctional mitochondria preventing their pathological accumulation and contributing to cellular homeostasis. We previously identified a novel chemical tool (hereafter referred to as PMI), which drives mitochondria into autophagy without collapsing their membrane potential (ΔΨm). PMI is an inhibitor of the protein-protein interaction (PPI) between the transcription factor Nrf2 and its negative regulator, Keap1 and is able to up-regulate the expression of autophagy-associated proteins, including p62/SQSTM1. Here we show that PMI promotes mitochondrial respiration, leading to a superoxide-dependent activation of mitophagy. Structurally distinct Keap1-Nrf2 PPI inhibitors promote mitochondrial turnover, while covalent Keap1 modifiers, including sulforaphane (SFN) and dimethyl fumarate (DMF), are unable to induce a similar response. Additionally, we demonstrate that SFN reverses the effects of PMI in co-treated cells by reducing the accumulation of p62 in mitochondria and subsequently limiting their autophagic degradation. This study highlights the unique features of Keap1-Nrf2 PPI inhibitors as inducers of mitophagy and their potential as pharmacological agents for the treatment of pathological conditions characterized by impaired mitochondrial quality control.

Association of a Platinum Complex to a G-Quadruplex Ligand Enhances Telomere Disruption.

Telomeres protect the ends of chromosomes against illegitimate recombination and repair. They can be targets for G-quadruplex ligands and platinum complexes due to their repeated G-rich sequences. Protection of telomeres is ensured by a complex of six proteins, including TRF2, which inhibits the DNA damage response pathway. We analyzed telomere modifications induced in cancer cells by the experimental hybrid platinum complex, Pt-MPQ, comprising both an ethylene diamine monofunctional platinum complex and a G-quadruplex recognition moiety (MPQ). Pt-MPQ promotes the displacement of two telomeric proteins (TRF2 and TRF1) from telomeres, as well as the formation of telomere damage and telomere sister losses, whereas the control compound MPQ does not. This suggests that the platinum moiety potentiates the targeting of the G-quadruplex ligand to telomeres, opening a new perspective for telomere biology and anticancer therapy. Interestingly, the chemotherapy drug cisplatin, which has no specific affinity for G-quadruplex structures, partially induces the TRF2 delocalization from telomeres but produces less telomeric DNA damage, suggesting that this TRF2 displacement could be independent of G-quadruplex recognition.

Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer's disease.

Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer's disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aß42 peptide, in correlation with Nrf2 activation. Comparatively, lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aß42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aß oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo.