Developing Fluorescent Sensors for the Bioimaging of Chelatable Iron(III) Ions

Iron is an essential element for the body and plays important roles in many metabolic processes. A transient labile iron pool (LIP) has been proposed to play key roles in cellular iron trafficking and metabolism. However, free iron ions (Fe3+ and Fe2+) in this pool are toxic and damaging to cells due to their involvement in the production of oxygen radicals. The damages may lead to aging and various diseases including stroke, cancer, and several neurological disorders like Parkinson’s disease, Alzheimer’s disease and atherosclerosis. Determination of free iron ions in cells may contribute to a better understanding of iron’s function and transport pathways under physiological and pathological conditions. Early methods to identify a labile and transient iron pool needed to disrupt cells. Over the past two decades, fluorescent sensors have emerged to visualize metal ions in living cells without any damage. To visualize intracellular iron ions, specific fluorescent sensors are needed. Even though fluorescent sensors have been applied into cells for two decades, only a few Fe3+-selective sensors are capable of cellular imaging with limited success. Chapter 1 introduces the biological background of iron as well as recently developed fluorescent sensors for iron detection. In chapter 2, a new rhodamine-based sensor, RPE, was synthesized and characterized by 1H NMR, 13C NMR, and MS. The sensor responds to Fe3+ via coordination induced fluorescent activation (CIFA) mechanism and gives a distinct rapid and reversible fluorescence response upon the alteration of intracellular Fe3+ levels with little interference from other biologically relevant metal ions. RPE can readily detect endogenous chelatable Fe3+ via a confocal microscope in live human SH-SY5Y cells at subcellular resolution in real time, with two labile Fe3+ pools being successfully located in mitochondria and endosomes/lysosomes in both untreated and Fe3+-loaded human SH-SY5Y cells for the first time. RPE is thus a promising tool for probing the cell biology of Fe3+. Sensors, such as RPE, which emit lights in visible range, suffer from some significant drawbacks, including intrinsic signal caused by auto-fluorescence, high light scattering, and poor light penetration when they are applied to biological tissues. To overcome these limitations, near infrared sensors can be used because they emit at longer wavelength so they display low autofluorescence background, deeper penetration to tissues and cause less damage to biological samples. In chapter 3, a heptacyanine based sensor, IRPE, was synthesized and characterized by 1H NMR, 13C NMR, and MS. IRPE showed selective response to Fe3+ and binds it in a 1:1 stoichiometry with an apparent binding constant 2.0×105 M−1 in ACN/HEPES (1/1 v/v) solution. The sensor displays a change in color and fluorescence upon the alteration of Fe3+ levels in solution with a reversible response and little interference with other biological relevant metal ions. IRPE is a good Fe3+-selective sensor but, cell studies showed that it was not capable of detecting free iron ions in cells. To get cell permeable and Fe3+-selective near infrared sensors, it was decided to change the strategy for near-infrared sensor design. In chapter 4, a Changsha based sensor, NIRh-Ac, was developed. The sensor gives a distinct rapid and reversible fluorescence response upon the alteration of intracellular Fe3+ levels with little interference from other biologically relevant metal ions. Confocal experiments showed that NIRh-Ac could readily detect exogenous chelatable Fe3+ in live human SH-SY5Y cells and live fibroblast cell (ws1) at subcellular resolution, with the chelatable Fe3+ pools located in mitochondria and endosomes/lysosomes for SH-SY5 cells and in mitochondria for ws1 cells. Kinetic experiments with the sensor provided a visual imaging of Fe3+ transport pathway in human fibroblast cells in real time, i.e., from endosomes to lysosomes and finally to mitochondria via a direct “Lyso-mito docking” mechanism, bypassing the cytosol. Studies using zebrafish clearly demonstrated the capability of the NIRh-Ac sensor in imaging Fe3+ in live animals. In chapter 5, another Changsha-based near infrared sensor, NRPA, was developed to detect endogenous Fe3+ ions in cells. It is a highly sensitive, highly selective, and reversible turn-on near infrared fluorescent sensor for Fe3+. The sensor gives a rapid and reversible fluorescence response upon the alteration of intracellular Fe3+ levels with little interference from other biologically relevant metal ions. Confocal imaging studies demonstrate that NRPA can readily detect endogenous free Fe3+ in live human SH-SY5Y cells and live fibroblast cell (ws1) at subcellular resolution, with the chelatable Fe3+ pools located in mitochondria and endosomes/lysosomes in SH-SY5 cells while in mitochondria only in ws1 cells. It was concluded that different cell lines store/handle iron in different ways. The ability of NRPA to detect endogenous free Fe3+ ions in zebrafish was also demonstrated and free Fe3+ ions were found located in liver/gall bladder of 6-days-old zebrafish. In chapter 6, highly sensitive, highly selective, and reversible turn-on near infrared Changsha-based fluorescent Fe3+-sensors, NRPK and NRP were described. The initial goal was to synthesize Fe(II)-selective fluorescence sensor; however, NRPK turned out to be a sensor for Fe (III). Both NRP and NRPK coordinate Fe3+ with O/N/N binding motif with 2:1 ratio. The NRPK appears not to use the carbonyl group linked to the pyridine ring for coordination. Cell imaging experiments with NRPK showed that it could readily detect endogenous free Fe3+ in live bovine aortic endothelial cells (BAEC) and human SH-SY5Y cells at subcellular resolution, with free iron (III) ions located in mitochondria and endosomes/lysosomes for both BAEC and SH-SY5 cells. Finally, NRPK demonstrated the ability to detect exchangeable free iron(III) in zebrafish in vivo. Finally, a novel sensor was developed to quantify the concentration of Fe3+ in the LIP and to confirm that the images observed by the sensors such as RPE, NIRh-Ac, NRPA and NRPK reflect the true locations of cellular chelatable Fe3+, not instead the locations of the sensors themselves. A ratiometric near infrared sensor, CR-PK, was presented in chapter 7.The CR-PK sensor shows NIR and visible emission in its spirolactam ring-open and closed forms, respectively. The reaction of CR-PK with Fe3+ leads to the ring opening of the spirocyclic moiety of CR-PK, causing a large red shift ~222 nm of the absorption band. Cell imaging experiments with CR-PK revealed that CR-PK is evenly distributed in the cells except the nuclei region; however, chelatable labile Fe3+ ions are located in certain organelles in live bovine aortic endothelial cells (BAEC), fibroblast (ws1) cells and human neuroblastoma cell (SH-SY5Y). The ratiometric sensor CR-PK enables the direct determination of endogenous labile Fe3+ concentration in the cells for the first time, with a value of~0.6 µM determined (0.43 ± 0.23 µM by Method 1 and 0.8 ± 0.28 µM by Method 2 ) for ws1 cells, ~1.78 µM determined (2.18 ± 0.35 µM by Method 1 and 1.38 ± 0.47 µM by Method 2 ) for BAEC cells, and ~3.05 µM determined (3.2 ± 0.63 µM by Method 1 and 3.1 ± 0.53 µM by Method 2 ) for SH-SY5Y cells. The various highly selective Fe3+ sensors developed in this work offer novel tools for molecular imaging of Fe3+ in live cells and live animals. The sensors, covering wavelength in the visible and near infrared regions with affinity to Fe3+ in micro to nanomolar levels, are ideal to image Fe3+ at subcellular resolution in real time, with the chelatable Fe3+ pools identified in various cell lines and live animals for the first time. Moreover, the ratiometric sensor enabled the determination of Fe3+ concentration in live cells for the first time. The Fe3+ sensors will contribute to a better understanding of the cell biology of iron and its related pathology and medical applications

A pH Stable Turn-on Fluorescent Sensor for Imaging Labile Fe3+ in Living Cells

Fluorescent sensors has received considerable interest in recent years because of its ability to provide visualized monitoring of very low concentrations together with the advantages of spatial and temporal resolution. Over the past two decades, several fluorescent sensors for iron (III) have been reported. However, the currently known fluorescent sensors that are capable of cellular iron imaging are largely limited to “turn-off” type, providing useful information but suffering from poor sensitivity, or interference from other metal ions. We have been developing rhodamine based turn-on fluorescent sensors. Here we report a new iron (III) sensor , Rh-PK, which is stable in low pH\u27s and is capable of detecting basal level Fe+3 in the human live cells at subcellular resolution

Prenatal multivitamin supplementation increases birth weight

Background: The aim of this study is to examine whether there is any positive impact of prenatal iron and vitamin supplementation on birth weight, and if this practice could be a cause of macrosomia.Methods: A total of 1,838 term pregnant women were included in this study and were divided into four groups: iron group (women having only iron supplementation), vitamin (women using only multivitamins), vitamin+iron (women using both iron and multivitamin preparations), and control group (women having neither iron nor vitamin supplements). Statistical analysis was performed to compare age, gravida, parity, hemoglobin, BMI and birth weight. Groups were compared in terms of macrosomia and correlation analysis carried out between demographics and birth weight.Results: The birth weight was significantly higher in the vitamin group and the vitamin+iron group than in the iron group and the control group. Vitamin supplementation increased the risk of macrosomia 3.9 times, while vitamin+iron usage increased the risk 4.8 times.Conclusions: Uncontrolled use of multivitamins and/or iron supplements may increase tendency for fetal macrosomia. The use of multivitamins and/or iron supplements by pregnant women must take into account maternal age, maternal diet and maternal BMI. Our findings support a reduction in the unnecessary use of vitamin and iron supplements in pregnancy, and a reversal of the trend to start vitamin and iron supplementation without any control. The increasing prevalence of obesity and excessive weight in pregnant women should be a warning against the uncontrolled usage of vitamins and iron during pregnancy

Improving the sensitivity of cochlear implant integrity testing by recording electrode voltages with surface electrodes

Introduction: Identification of faults with the internal, implanted, part of a cochlear implant presents a challenge for the cochlear implant community. Advanced Bionics Ultra V1 devices are vulnerable to moisture ingress, a hard failure, resulting in reduced volume and clarity for the recipient. The manufacturer uses a trans-impedance test “Electrical Field Imaging” to identify faulty Ultra V1 devices but reports the sensitivity of the test to be only 70–90%. Methods: In our clinic we performed Electrode Voltage measurements with surface electrodes and have compared the two tests. Electrical Field imaging and Electrode Voltage (EV) measurements were available for 65 devices. Surface electrodes were attached to the earlobes and forehead and potentials measured in three montages: ipsilateral earlobe and forehead, contralateral earlobe and forehead, and both earlobes; voltages were extracted and relative voltages across the array were calculated. Results: Relative EV were compared for the two earlobes montage and fitted to a third order polynomial function. A new criterion for identifying faulty devices was derived, with a deviation of N = 15) had a normal electrical field imaging test, whilst 17/50 devices which were abnormal had normal electrical field imaging and 33/50 which were abnormal had abnormal electrical field imaging. Discussion: The REVs test was well-tolerated and carried out in a routine cochlear implant clinic. Together with test sensitivity and reliability this may make it a new routine assessment tool to aid in distinguishing hard and soft failures

The Resource Curse and Rentier States in the Caspian Region : A Need for Context Analysis

Although much attention is paid to the Caspian region with regard to energy issues, the domestic consequences of the region’s resource production have so far constituted a neglected field of research. A systematic survey of the latest research trends in the economic and political causalities of the resource curse and of rentier states reveals that there is a need for context analysis. In reference to this, the paper traces any shortcomings and promising approaches in the existent body of literature on the Caspian region. Following on from this, the paper then proposes a new approach; specifically, one in which any differences and similarities in the context conditions are captured. This enables a more precise exploration of the exact ways in which they form contemporary post-Soviet Caspian rentier states.Obwohl der Region am Kaspischen Meer im Zuge von Energiediskursen große Aufmerksamkeit zuteil wird, stellen die innerstaatlichen Folgen der Ressourcenproduktion in der Region ein bislang vernachlässigtes Forschungsfeld dar. Ein systematischer Überblick über die jüngsten Forschungstrends zu wirtschaftlichen und politischen Kausalzusammenhängen des Ressourcenfluchs und zu Rentierstaaten offenbart die Notwendigkeit von Kontextanalysen. Hierauf Bezug nehmend, analysiert der Aufsatz sowohl die Mängel als auch viel versprechende Ansätze in der betreffenden Literatur zur Region am Kaspischen Meer. Der Aufsatz stellt letztendlich einen neuen Ansatz vor, der Unterschiede und Gemeinsamkeiten in den Kontextbedingungen erfasst, um zu erforschen, wie diese die gegenwärtigen post-sowjetischen Rentierstaaten in der Region am Kaspischen Meer tatsächlich prägen

30-Day morbidity and mortality of bariatric metabolic surgery in adolescence during the COVID-19 pandemic – The GENEVA study

Background: Metabolic and bariatric surgery (MBS) is an effective treatment for adolescents with severe obesity. Objectives: This study examined the safety of MBS in adolescents during the coronavirus disease 2019 (COVID-19) pandemic. Methods: This was a global, multicentre and observational cohort study of MBS performed between May 01, 2020, and October 10,2020, in 68 centres from 24 countries. Data collection included in-hospital and 30-day COVID-19 and surgery-specific morbidity/mortality. Results: One hundred and seventy adolescent patients (mean age: 17.75 ± 1.30 years), mostly females (n = 122, 71.8%), underwent MBS during the study period. The mean pre-operative weight and body mass index were 122.16 ± 15.92 kg and 43.7 ± 7.11 kg/m2, respectively. Although majority of patients had pre-operative testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (n = 146; 85.9%), only 42.4% (n = 72) of the patients were asked to self-isolate pre-operatively. Two patients developed symptomatic SARS-CoV-2 infection post-operatively (1.2%). The overall complication rate was 5.3% (n = 9). There was no mortality in this cohort. Conclusions: MBS in adolescents with obesity is safe during the COVID-19 pandemic when performed within the context of local precautionary procedures (such as pre-operative testing). The 30-day morbidity rates were similar to those reported pre-pandemic. These data will help facilitate the safe re-introduction of MBS services for this group of patients

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely