A marked surge in the blood reticulocyte count was observed in paraquat treated mice that also subsided by 28th day. Young erythrocytes in circulation were randomly eliminated from blood circulation in paraquat treated mice and a significant elevation in the level of reactive oxygen species (ROS) was also observed maximally the erythrocytes of this age group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were identified and enumerated flow cytometrically based on their expression of Ter119 and transferrin (CD71) receptor. Proliferative activity of erythroid cells, their relative proportion as well as their absolute numbers fell significantly in bone marrow of paraquat treated mice but all these parameters were significantly elevated in spleens of paraquat treated mice. These changes were essentially restricted to the cells belonging to the two earliest stages of erythroid differentiation. Taken together our results indicate that paraquat treatment causes a transient anemia in mice resulting from random elimination of young circulating erythrocytes as well as depressed erythropoietic activity in bone marrow. Spleen erythropoietic activity however was elevated in paraquat treated mice.Paraquat a widely used herbicide causes a variety of toxic effects on humans and animals. The present study is focused on the interaction of paraquat with the mouse erythroid system. Administration of paraquat  induced a significant fall in blood erythrocyte count on 7, 14, and 21 day time points but the erythrocyte count reverted back to normal by 28 Paraquat  was first synthesized in 1880 but its action as a potent herbicide was discovered only in 1955 Ingestion of paraquat causes liver, lung, heart, and kidney failure within several days to several weeks Erythropoiesis is a multistep process that starts with the commitment of pluripotent hematopoietic stem cells (HSCs) progeny into erythroid line of differentiation Results of our present study show that the administration of paraquat results in an acute yet transient anemia in mice. Young erythrocytes that are not eliminated from circulation in control mice are eliminated at a significant rate in paraquat treated mice. Depressed proliferative activities and elevated cell death was observed in cells of erythroid lineage in the bone marrow of paraquat treated mice. In contrast, spleen erythropoietic activity was found to be significantly enhanced after paraquat administration suggesting that spleen may play a significant role in enhanced compensatory erythropoietic activity in paraquat treated mice.male mice  were used throughout this study. Animals were bred and maintained in microbes free environment in the animal house facility at Jawaharlal Nehru University (JNU), New Delhi or obtained from the National Institute of Nutrition, Hyderabad. The animals were housed in positive-pressure air conditioned units  and kept on a 12 h light/dark cycle. Water and mouse chow were provided ad libitum. All the experimental protocols were conducted strictly in compliance with the Guidelines notified by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India . The study was duly approved by JNU Institutional Animal Ethics Committee (IAEC Approved Project Code: 5/2010). All mice were randomly assigned to experimental groups. Experiments were designed so as to use the minimum number of mice. Numbers of mice used in various experiments are provided in figure legends. Mice were administered the required dose of paraquat by intra-peritoneal injections on alternate days. For the analysis of blood erythrocytes, 25 to 50 µl blood samples were taken weekly from tail-vein at different indicated time points as per prescribed CPCSEA guidelines. For deriving bone marrow and spleen cells, mice were euthanized by cervical dislocation before the organs were dissected out. All efforts were made to minimize pain.Inbred C57BL/6 2DCFDA) was purchased from Molecular Probes . Mouse Easy Sap biotin selection kit was procured from Stem cell technology (USA). Revert Aid First strand cDNA synthesis kit was from Thermo Scientific (India). Power SYBR green PCR Master mix was from Applied Biosystem . Fetal bovine serum was obtained from Hyclone . TRI reagent, RPMI, HEPES, Thiazole Orange (TO), Dimethylformamide (DMF), Paraquat and other analytical reagents were from Sigma-Aldrich (India).Biotin-X-NHS (N-hydroxysuccinimide ester of biotin) was obtained from Calbiochem . Streptavidin Allophycocyanin (SAv-APC), rat anti-mouse Ter-119-APC, rat anti-mouse Ter-119-FITC, anti-mouse CD16/CD32 were from BD Biosciences . Anti-mouse CD71-PE, anti-mouse CD71-biotin, Annexin-V-FITC, anti-mouse IgG1κ-PE, anti-mouse IgG2bκ-APC, anti-mouse IgG2bκ-FITC, 7-Aminoactinomycin D (7-AAD) were procured from e-biosciences . 5 (and 6) chloromethyl-2, 7 dichloro-dihydro-fluorescein diacetate  (three daily intravenous (i.v.) injections of 1 mg BXN dissolved in 20 µl of dimethyl formamide (DMF) and 250 µl of phosphate buffered saline). Mice are rested for 5 days and fresh erythrocytes that entered the blood after the first biotinylation step were biotinylated at a lower intensity by a single i.v injection of 0.6 mg of BXN dissolved in 12 µl of DMF and 250 µl of PBS. At any time point after the second biotinylation step, biotin intensity on circulating erythrocytes could be analyzed flow cytometrically after staining the cells with streptavidin coupled to an appropriate fluorochrome. Biotinnegative erythrocytes in circulation would represent fresh erythrocytes released in blood after the second biotinylation step. Biotinl°w erythrocytes would represent the cohort of erythrocytes released in blood between the first and the second biotinylation steps, and biotinhigh erythrocytes would represent the population of residual erythrocytes that were present in the blood at the time of first biotinylation step Rationale and details of the DIB technique, its use in studying erythrocyte turnover and age related changes in erythrocytes Paraquat dichloride hydrate  was freshly dissolved in PBS for each injection. Mice were administered repeated doses of paraquat (10 mg/kg of body weight) intra-peritonealy on alternate days 2DCFDA stain (5 µM) in the dark for 30 minutes at 37°C in an atmosphere of 5% CO2 in air. The oxidative conversion of CM-H2DCFDA to its fluorescent product by ROS was measured immediately by flow cytometry.Intercellular ROS levels were assessed as described before + reticulocytes were sorted by using Easy Sap biotin cocktail and magnetic beads reagent according to manufacturer’s instruction (Stem cell technology) +, CD71+ cells). Total RNA in sorted reticulocytes was extracted by TRI reagent (Sigma) and reverse transcribed into cDNA by utilizing the First Strand cDNA synthesis kit  as per manufacturer’s instructions. Relative levels of SOD1 mRNA in reticulocytes were estimated by quantitative RT-PCR (Primers 5′-GAGACCTGGGC AATGTGACT-3′ and 5′-TTGTTTCTCATGGACCACCA-3′) as described before −ΔΔCT method Reticulocytes within the erythrocyte population were identified by CD71 staining. Blood cells were stained with biotinylated anti-mouse CD71 monoclonal antibody (3 µg/ml) in PBS supplemented with 2%FBS for 20 min at 4°C, and CD71Mouse blood was collected in PBS containing 5 mM EDTA and washed 3 times with ice cold saline containing HEPES buffer  and 1% FBS. Biotin labeled erythrocytes were stained with streptavidin-FITC or streptavidin-APC and analyzed on a flow cytometer as described before In some experiments, reticulocyte population in blood was enumerated flow cytometrically as described before 6 cells in 50 µL of PBS+2%FBS) for 10 min followed by staining with anti-CD71-PE and anti Ter-119-APC for 20 minute at 4°C For enumerating erythroid cells at different stages of differentiation in bone marrow and spleen, freshly prepared single cell suspensions from bone marrow or spleen were first incubated with anti-CD16/32 antibody  for immediate flow cytometric analysis.For cell cycle study, methodology of Marinkovic p<0.05 was considered significant.Statistical analysis was carried out by using Sigma plot software. Standard t-tests were used to calculate the significance levels between groups. While the occurrence of anemia in subjects exposed to paraquat has been documented in vivo biotinylation (DIB technique) of circulating erythrocytes (24). In this technique a cohort of erythrocytes entering blood circulation within a defined window of 5 days can be tracked as it ages in real time in blood circulation. This cohort is identified as erythrocyte subpopulation in box “Y” (biotinl°w subpopulation of blood erythrocytes) in high subpopulation of erythrocytes) represent older erythrocytes of age greater than that of the biotinl°w cohort of erythrocytes being tracked, and erythrocytes in box “Z” (biotinnegative subpopulation of erythrocytes) represent younger erythrocytes of age lower than the tracked cohort of erythrocytes (l°w subpopulation from 14.86% in control to 8.44% (43% decline) in paraquat treated mice indicates that 14 days after the paraquat treatment, the cohort of erythrocytes that entered the blood immediately before paraquat treatment started, had declined 43% more as compared to the similar cohort in control mice  encoded in SOD1 gene, is an enzyme that deactivates superoxide radicals, which is an important component of ROS. Reduced SOD levels have been shown as a causative factor in anemia med CD71high), early basophilic erythroblast (Terhigh CD71high) (erythroblast A), late basophilic, polychromatophilic erythroblast and orthochromatophilic erythroblast (Terhigh CD71med) (erythroblast B) and orthochromatophilic erythroblast with mature erythrocytes (Terhigh CD71l°w) (erythroblast C). Total erythroid population in bone marrow can be enumerated by setting an inverted L-shaped gate in flow cytometric histograms that include all the above mentioned four stages of erythroid differentiation Bone marrow and spleen are two prime erythropoietic sites in adult mice To investigate the effect of paraquat treatment on erythropoiesis, we examined the relative proportions as well as absolute recoveries of erythroid cells in bone marrow and spleen of control and paraquat treated mice. Results of a representative experiment show that 30.9% of the cells in control bone marrow belonged to erythroid lineage. In paraquat treated mice, the proportion of erythroid cells fell to 17.97%, suggesting a significant fall in erythropoietic activity in bone marrow . In contIn contrast to the observation in bone marrow, the proportion of erythroid cells increased significantly (range 21% to 41% increase) in spleens of paraquat treated mice especially on day 7, 14 and 21 day of treatment . HoweverSince there are four well defined stages of erythroid differentiation in bone marrow and spleen + erythroid cell populations. Typical results of the cell cycle analysis are provided as supplementary data . Cell cycle analysis was carried out on gated Ter119ary data . Combine+ erythroid cells in bone marrow and spleen in control and paraquat treated mice. Cells were stained with Ter119 antibody as well as with Annexin-V-FITC and 7-AAD. Typical results of the analysis of apoptotic cells is given as supplementary data  technique that allows us to study erythrocyte cohorts of defined age groups in blood circulation An important aim of this study was to find if the age of erythrocytes in circulation is a factor in determining their elimination in paraquat treated mice. We have recently developed a double We found a significant increase in the levels of reactive oxygen species (ROS) in erythrocytes from paraquat treated mice. An increase in ROS levels coupled with the fact that erythrocytes lack nucleus and therefore robust self-repair mechanisms; suggest that increased ROS could be a contributory factor in the induction of anemia in paraquat treated mice. Further supportive evidence of an involvement of ROS in paraquat induced anemia is our finding of a significant decline in the mRNA levels for superoxide dismutase (SOD) enzyme in reticulocytes from paraquat treated mice. SOD deactivates superoxide radicals that constitute an important component of ROS and a decline in SOD expression in reticulocytes  and paraquat treated (panel B) mice. Similar data for spleen cells is shown in panels C and D. Percentage values for cells in Go//G1, S and G2/M phases are given.(TIF)Click here for additional data file.Figure S2Paraquat induced apoptotic and necrotic changes in bone marrow and spleen erythroid cells. Mice were treated with repeated doses of paraquat. Bone marrow and spleen cells were harvested after 14 days of treatment and stained with anti-mouse Ter 119-APC, along with Annexin-V FITC and 7-AAD followed by flow cytometric analysis. Apoptotic cells were quantified in Ter119 gated population on the basis of Annexin V and 7-AAD staining. Panels A and B show the representative flow cytometric histograms for bone marrow cells derived from control and paraquat treated mice. Similar data for spleen cells is shown in panels C and D. Values in different quadrants represent percentage of cells. Apoptotic cells are in top right quadrants and the necrotic cells in lower right quadrants.(TIF)Click here for additional data file.


Microcavities filled with biologically produced green fluorescent protein show polariton condensation at room temperature. Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing. Q microcavities without damaging the organic emitter contained inside has necessitated elaborated device fabrication processes. Finally, the characteristic two-threshold behavior expected for a polariton laser (where the second threshold indicates the onset of photon lasing) has not been observed in organic devices so far.Strong light-matter coupling occurs when interactions between the exciton reservoir of an active medium and a surrounding confined light field—typically generated by an optical microcavity—are strong enough to result in the formation of bosonic coupled light-matter particles, known as cavity polaritons . The emission of these hybrid bioinorganic devices shows a clear two-threshold behavior, corroborating the buildup of a polariton condensate at low excitation densities and the onset of photon lasing (with simultaneous transition to the weak-coupling regime) at higher excitation densities. Both PC and photon lasing are achieved with conventional nanosecond excitation, eliminating the need for ultrashort pump pulses.Here, we demonstrate polariton lasing from simple laminated high-There is growing interest in understanding how biomaterials affect light at the quantum level, for example, with respect to coherence and entanglement in photosynthetic complexes  E0,TDAF = 0.037 mJ/cm2, the brightness of the TDAF sample began to increase more slowly (∝ E1/2). This square root dependence is a fingerprint of bimolecular exciton-exciton annihilation being the dominant channel of exciton decay . Using the critical fluence for each material and a rate equation model, we estimate effective annihilation rate constants of kXX,eGFP = 2.4 × 109 s−1 and kXX,TDAF = 6.1 × 1010 s−1 for eGFP and TDAF, respectively (section S1), indicating that the high exciton densities required for PC are achieved more readily with eGFP. The relatively low annihilation rate constant also implies that large exciton densities may be achieved in eGFP films using nanosecond, instead of picosecond or femtosecond, pumping.To investigate the performance of FPs at high exciton concentrations, we compared the fluorescence of thin films of eGFP and of 2,7-bis-9,9-di(4-methylphenyl)fluorine (TDAF), one of the materials in which PC of organic polaritons was recently achieved  . HoweverQ factor of the involved cavity modes and increase the photonic character of the coupled cavity polaritons , which then also reduces the polariton linewidth.We next fabricated laminated microcavities composed of an eGFP film  sandwiched between two dielectric Bragg mirrors . The lamn) but also the first Bragg mode  (BM) of the dielectric mirrors. For the microcavity containing eGFP with high water content, the cavity modes and the Bragg mode overlap spectrally (at certain thicknesses). By contrast, a clear splitting occurs between all observed modes and for all thicknesses for the eGFP film with low water content. This avoided crossing is a clear signature of strong coupling between eGFP excitons and cavity modes. We attribute the difference between the two samples to the higher concentration of eGFP fluorophores in the film with low water content. In “traditional” polariton microcavities with only one exciton and one photon mode, anticrossing is expected between the upper and the lower polariton branch, as well as between the polariton branches and uncoupled exciton and photon modes. Only the latter is present here because of the multimodality of both the photonic and electronic systems. Fitting a coupled oscillator model to the experimental data for the low–water content sample yields coupling strengths of V1 = (97 ± 8) meV and V2 = (46 ± 5) meV for the X1 and X2 transitions, respectively (section S2).We found that the density of eGFP within the active region of the microcavity is crucial to realizing strong cavity-photon interaction. Reflectance measurements of microcavities containing eGFP with high and low water content  are shown in n with strongly decreasing intensity toward higher emission angles, following a Boltzmann occupation along the polariton dispersion (section S4), thus showing the absence of any polariton bottleneck  shifted to higher energies, (ii) increased nonlinearly in intensity  using nanosecond pulses. Whereas the exciton lifetime of eGFP is in the few-nanosecond range  At the same time, the emission from the condensed cavity polaritons remains energetically below the expected dispersion of uncoupled cavity photons  and photon (P2) lasing  gives insight into the wavelength dependence of the thresholds for polariton  lasing . We obseThe compatibility with nanosecond optical pumping and the simplicity of our lamination approach represent major improvements over previous organic polariton lasers. They bring PC and its remarkable properties, such as low-threshold coherent light generation and superfluidity, within easier reach and represent a step toward continuous-wave polariton lasing at room temperature. To the best of our knowledge, the two-threshold behavior observed here has not been reported for an organic system so far because of a combination of photobleaching and exciton-exciton annihilation at high excitation densities (6 tag for protein purification. Escherichiacoli strain BL21(DE3)pLysS (Novagen) was transformed with the construct using LB medium with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml) for selection. Cells were grown at 30°C until OD600  of 0.5 to 0.6 and harvested 4 hours after protein expression was induced with isopropyl-β-d-thiogalactopyranoside . Cells were resuspended in buffer  and disrupted by sonification and treatment with lysozyme, and eGFP was purified using His•Bind Resin (Novagen). After elution, the protein-containing fraction was dialyzed against phosphate-buffered saline twice using a Slider-A-Lyzer Dialysis cassette  (Thermo Fisher Scientific). Subsequently, eGFP was filtered and centrifuged to remove buffer salts and increase protein concentration (to up to 220 g/liter). Concentrated solutions (~100 μl) were spin-coated (1000 rpm for 60 s) onto dielectric mirrors  designed for peak reflectance (R ≥ 99.995%) at a wavelength of 532 nm and consisting of 14 pairs of alternating SiO2 (73-nm)/Ta2O5 (59-nm) layers. The structure was capped with an identical mirror on top to form the microcavity. Subsequently, the eGFP solution dried out and left a solid eGFP film with a nearly constant thickness of around 500 nm. Microcavities were characterized directly after spin coating and again after the drying process. In this way, the same sample with different water content of the eGFP film inside the cavity was investigated. The manual capping yielded a gradient in cavity thickness, spanning from 1500 to 4500 nm, which enabled scanning through different cavity modes by translating the sample. The theoretical quality factors of the photonic modes reach up to Q = 50,000. The thin films of eGFP for fluorescence characterization were fabricated on glass substrates in the same manner as described above. TDAF (Lumtec) was used as a representative example of a synthetic organic polariton laser material. Thin films of TADF were deposited by thermal evaporation under high vacuum.The open reading frame of eGFP was polymerase chain reaction–amplified and cloned into vector pET23b (Novagen) using standard molecular biology methods  covering an angular range of ±25°. The reflected light was collected through the same objective, dispersed by a 500-mm imaging spectrograph with a spectral resolution down to 0.03 nm, and imaged onto a cooled electron-multiplying charge-coupled device array detector. For measurements of the microcavity emission, the structure was excited by a pulsed, wavelength-tunable optical parametric oscillator system tuned to 460 nm  using the same optical setup as for the reflectance measurements. The pump laser is slightly elliptic and was focused down to an 8-μm spot  on the sample. The same system was also used for the fluorescence measurements of eGFP and TDAF on glass substrates.EX1 at 2.53 eV, its less pronounced sideband transition EX2 at 2.67 eV, and the uncoupled photon mode energies EnPh,, as well as the coupling strengths V1 and V2 to the transitions EX1 and EX2, respectively. Because of the simultaneous coupling to different cavity modes CMn and the Bragg mode BM, we treat the light field as a nonpure state (superposition of different modes). For most spectra, we achieved a quantitative agreement between experiment and theory by taking 10 photon modes close to the exciton resonances into account. The uncoupled cavity dispersions were determined by transfer matrix calculations (section S2) using a background refractive index of 1.51 for the spin-coated eGFP thin film.To model the experimentally observed dispersions of cavity photons and polaritons, and to determine the coupling strengths between eGFP excitons and cavity photons, we calculated the eigenstates of a coupled oscillator matrix that accounts for several cavity modes strongly interacting with two main excitonic transitions of the eGFP fluorophore. The matrix reads as follows


Chronic stress or long-term administration of glucocorticoids disrupts the hypothalamus-pituitary-adrenal system leading to continuous high levels of glucocorticoids and insulin resistance (IR). This pre-diabetic state can eventually develop into type 2 diabetes mellitus and has been associated with a higher risk to develop depressive disorders. The mechanisms underlying the link between chronic stress, IR and depression remains unclear. The present study aimed to establish a stress-depression model in mice to further study the effects of stress-induced changes upon insulin sensitivity and behavioural consequences. A pilot study was conducted to establish the optimal administration route and a pragmatic measurement of IR. Subsequently, 6-month-old C57BL/6NCrl mice were exposed to long-term oral corticosterone treatment via the drinking water. To evaluate insulin sensitivity changes, blood glucose and plasma insulin levels were measured at different time-points throughout treatment and mice were behaviourally assessed in the elevated zero maze (EZM), forced swimming test (FST) and open field test to reveal behavioural changes. Long-term corticosterone treatment increased body weight and decreased insulin sensitivity. The latter was revealed by a higher IR index and increased insulin in the plasma, whereas blood glucose levels remained unchanged. Corticosterone treatment induced longer immobility times in the FST, reflecting depressive-like behaviour. No effects were observed upon anxiety as measured in the EZM. The effect of the higher body weight of the CORT treated animals at time of testing did not influence behaviour in the EZM or FST, as no differences were found in general locomotor activity. Long-term corticosterone treatment via the drinking water reduces insulin sensitivity and induces depressive-like behaviour in the C57BL/6 mouse. This mouse model could thus be used to further explore the underlying mechanisms of chronic stress-induced T2DM and its association with increased prevalence of major depressive disorder on the short-term and other behavioural adaptations on the longer term. Chronic stress or long-term glucocorticoid administration, as well as (genetic) impairments in HPA-axis or corticoid receptor function are proposed to participate in the aetiology and progression of many health problems, including psychiatric disorders such as major depression and anxiety. Numerous studies have documented hypercortisolaemia, HPA-axis hyperactivity and reduced stress responsivity in patients with depression One of the major metabolic effects of chronic hypercortisolaemia and a key feature in the development of the metabolic syndrome is insulin resistance (IR), a condition characterized by persistent high levels of peripheral glucose (hyperglycaemia) and insulin (hyperinsulinaemia) due to a reduced ability of insulin to lower blood glucose levels by uptake into muscle tissue (insulin insensitivity) In mice, long-term daily injections with corticosterone induces depressive-like behaviour and has thus been proposed as a reliable mouse model for studying the implication of stress and glucocorticoids in depression et al. Virgin female C57BL/6NCrl mice  were crossed with male APPswe/PS1ΔE9 mice  at age 8–12 weeks as described by Sierksma Experimental protocols were reviewed and approved by the local ethical Committee on Animal Experimentation of the Maastricht University  and met Dutch governmental guidelines. Throughout all experiments the principles of laboratory animal care were carefully followed in accordance with the official codes of practice guidelines  and all efforts were made to minimize the number of mice used and their suffering.ad libitum throughout the experiments, unless otherwise stated. Animal cages were supplied with paper rolls and tissues to enrich environmental conditions. Clean cages were provided on a weekly basis and mice were weighed weekly and prior to oral glucose administration (pilot study). Drinking water consumption was measured by weighing the bottles before and after refreshing them.Mice were individually housed on sawdust bedding in standard Macrolon cages and placed in an individually ventilated cage (IVC) unit, regulating temperature (±20°C) and humidity (40–60%). They were further maintained on a reversed 12∶12-hr light-dark schedule (lights on from 18∶00 h−06∶00 h) and continuous background noise was provided by a softly playing radio. Food  and tap water were available To facilitate environmental adaptation, mice were not treated or handled until several weeks after single housing on a reversed day-night cycle. During this initial adaptation period, mice were additionally habituated to general handling procedures, including weighing. For the pilot study, mice were also familiarized to oral  administrations by gavage for subsequent oral glucose administration procedures. At all times, blood samples were taken by means of saphenous vein puncture, as this method has been proven to be appropriate for repeated sampling in the same animal in a quick and painless manner and without the requirement of anesthesia Behavioural testing for the main study did not start until 6 weeks into CORT treatment, at approximately 7.5 months of age. This was done deliberately, to make potential future comparisons with the behavioural and cognitive effects in the APPswe/PS1ΔE9 possible. Testing was always done in the (active) dark phase of the rodent day-night cycle. Mice were initially exposed to the elevated zero maze (EZM), followed by the forced swim test (FST) and open field test (OF), with a three-week and one-week interval, respectively  corticosterone (CORT) was used, obtained from SIGMA-ALDRICH . For the pilot study, mice were administered with either CORT  or vehicle  via daily subcutaneous (s.c.) injection or received CORT  or vehicle  in the drinking water for a total amount of 4 weeks. As CORT is hydrophobic, it was dissolved in ethanol and added to the drinking water to make a 1% ethanol solution. As ethanol is not an appropriate solvent for injectables, CORT was dissolved in propylene glycol and further diluted with saline for the s.c. injections. The vehicle injection group was administered with a 50% propylene glycol solution in saline (5 ml/kg). Injections were alternated daily between left or right flank or over the shoulders (into the loose skin over the neck) to avoid discomfort produced by the procedure or injection-site infection/local skin reaction. Potential interfering ethanol effects in the vehicle drinking water group compared to the other 3 experimental groups were avoided by providing all mice with 1% ethanol in the drinking water.In the main study, CORT  or vehicle  was administered via the drinking water for a total time of 12 weeks. Water bottles containing CORT were covered with tinfoil to avoid light-induced degradation of the compound and water quantity was monitored several times throughout the week. When calculating weekly water intake, any water spillage when removing bottles from cages was systematically taken into account by applying a correction factor. This factor was calculated by taken the mean spillage of water after taking the bottle out of the cage for 7 times in a row.The effects of CORT treatment (daily s.c. injection versus drinking water) on insulin sensitivity were evaluated by means of oral glucose tolerance tests (OGTT). An OGGT consists of assessing the efficacy of glucose clearing by insulin in the blood by measuring blood glucose and/or plasma insulin levels at different time points after an oral (body weight-matched) dose of glucose. Mice were fasted for 6 h after which a glucose solution (D-(+)-Glucose, SIGMA-ALDRICH, Zwijndrecht, The Netherlands) of 250 mg/ml was administered orally by gavage (2 g/kg) as derived from Adrikopoulos et al. et al. in vivo insulin sensitivity. Based upon fasting blood glucose and plasma insulin levels , the following formula is used: HOMA-IR = fasting [insulin(mIU/l)]*fasting [glucose(mmol/l)]/22.5. Lee et al. The homeostasis model assessment of insulin resistance index (HOMA-IR), as described by Matthews Based upon the results of the effects of CORT treatment via daily s.c. injection versus drinking water upon body weight, glucose tolerance and insulin sensitivity, it was decided to rely on the HOMA-IR index for insulin sensitivity measurements in the main study. Baseline blood glucose and plasma insulin levels were measured at 3 different time-points throughout the main study, namely prior to the start of CORT treatment  and 4 (t1) and 12 weeks (t2) into CORT treatment. Blood glucose and plasma insulin levels were used to calculate the HOMA-IR index as described earlier (see The elevated zero-maze test (EZM) is a modification of the elevated plus-maze and models anxiety-like behaviour in rodents A modified forced swimming test (FST) consisted of exposing the animals to an inescapable water stress, using the total time spent in an immobile position as a measure of their level of behavioural despair The cylinders were positioned on top of a light box to create optimal illumination conditions for video tracking. A camera was placed 75 cm above the light box and connected to a video tracking system , which allowed automated recording of the total distance moved. Total immobility time for each mouse was scored manually by an independent researcher blind for experimental conditions.The open field (OF) was originally designed to simultaneously measure locomotion, exploration and anxiety by exposing mice to a novel and open space The equipment was made of acrylic and consisted of a grey square arena (50×50×25 cm), subdivided by grey walls (height: 25 cm) into 4 separate arenas of 25×25×25 cm. Four mice were tested in parallel by placing them in the centre of the arenas, allowing them to move around freely for a total time of 20 min. After each 20 min session, faecal boli were counted per arena/mouse and arenas were subsequently thoroughly cleaned with 70% alcohol. Sessions were run under dimmed lightning conditions and videotaped from above by a camera connected to a video tracking system  which allowed automated tracking of the total distance moved for each mouse separately.t-tests were performed to analyze differences between treatment conditions per time-point and the effects upon behaviour. Where appropriate, a post hoc Bonferroni test was used to further characterize the treatment effects.In the pilot study, changes in body weight and glucose tolerance over time were analyzed by means of a three-way analysis of variance (ANOVA) with Treatment and Administration Route as between-subject factors and Time as within-subject variable. In all other cases, changes over time were analyzed by means of two-way ANOVA with Treatment as between-subject factor and Time as within-subject variable. Separate one-way ANOVAs or independent Student’s P<0.05. All data are represented as mean ± standard error of the mean (S.E.M.). Analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (IBM SPSS Statistics version 20). GraphPad Prism, version 4.02 for Windows was used for preparing the graphs.In all cases the acceptable level of significance was set at F = 18.19; P<0.001]. However, changes over time depended on treatment  and route of administration . Moreover, a significant time*treatment*route interaction was found . An overall treatment effect  and effect of administration route  was revealed, however, no significant treatment*route interaction was observed .Significantly increased body weights were found over the course of 6 weeks  and 2 weeks  into CORT treatment. Significantly increased body weights were found after 3 weeks CORT treatment body weights were significantly increased  with higher body weights in the CORT drinking water condition compared to the vehicle drinking water group and the group receiving CORT via injection as revealed by post hoc analyses. The same was true at the end (after 4 weeks) of CORT treatment  with post hoc analyses showing significantly higher body weights in mice receiving CORT via the drinking water compared to mice receiving daily CORT injections. Only a trend was found toward increased body weights in the mice receiving CORT through drinking water compared to their vehicle counterparts (P = 0.07). One week after treatment cessation (week 5) body weights of mice receiving CORT via drinking water were still significantly higher compared to all other groups . Two weeks after treatment cessation body weights were still significantly increased , however, post hoc analyses revealed increased body weights only in the CORT drinking water group compared to the vehicle injection group. At this time point, no differences were found between the CORT drinking water and CORT injection condition or between the CORT drinking water and vehicle drinking water group  = 4.77; P<0.01]. In addition, a tendency towards a time*treatment interaction effect  and time*treatment*route interaction effect  was observed. Subsequent one-way ANOVA for detailed analysis of treatment effects per time-point reveals a trend towards impaired glucose clearance 30 min after oral glucose administration in the CORT via drinking water condition compared to the other treatment groups (P = 0.094). During OGTT2, only a significant time*treatment interaction  was revealed. Overall treatment effects upon glucose tolerance were not found during any of the OGTTs (see Glucose tolerance prior to the start of CORT treatment (OGGT0) is not depicted in a graph as it involves one baseline measurement of all mice together. Directly after 4 weeks CORT treatment (OGGT1), glucose tolerance differed over time, depending on route of administration . Treatment cessation led to a normalisation as no differences were found in HOMA-IR indices 1 (t2)  and 2 weeks after the end of CORT treatment (t3)  (Although no effects were found of CORT treatment upon fasting blood glucose and plasma insulin levels (independent of administration route), a significant higher HOMA-IR index in the CORT drinking water group compared to the vehicle drinking water group directly after the 4 weeks treatment period (t1) was found  .F = 21.40; P<0.001], but not for both groups (CORT versus vehicle) similarly . An overall treatment effect revealed that mice receiving CORT via the drinking water gained significantly more weight compared to the control vehicle group . Separate t-tests per week showed increased body weights in the CORT treated mice at all time points , except for week 2, which resulted in a trend towards increased body weight .Body weight see  significF = 4.06; P<0.001] and differently depending on treatment condition . However, no overall treatment effect was found . When analyzing differences between the treatment groups per time point, separate t-tests revealed significant higher amounts of water intake only at week 1 , 2  and 4  in the CORT treated animals  = 30.84; P<0.001], but differently depending on treatment condition . However, an overall treatment effect was not found, but did result in a trend towards increased blood glucose levels in the vehicle treated animals at 12 weeks .F = 3.06; n.s.] and HOMA-IR index  did not change over time, but overall treatment effects were found  = 39.19; P<0.001] and , respectively). Separate t-tests for treatment effects per time-point revealed significantly increased plasma insulin levels in CORT treated mice compared to the vehicle group after both 4  and 12 weeks  of CORT treatment and similar effects were found for the HOMA-IR index after both 4  and 12 weeks  of CORT treatment.Plasma insulin levels  in the EZM. However, the percentage of time spent in the open arms was not different between treatment conditions   = 3.37; P<0.01], whereas body weight at time of testing was significantly higher . In addition, CORT treated mice showed more depressive-like behaviour as was reflected upon by the significantly increased immobility times in this group   = 0.55; n.s.], whereas mean body weight of the CORT treated mice, like during the FST, was significantly higher compared to the vehicle group . Moreover, the amount of faecal boli was significantly higher in the CORT treated animals  (see For the OF, no effects of CORT treatment upon total distance moved were found [001] see .The present study aimed to establish a stress-IR-depression model in the mouse and specifically in the C57BL/6NCrl mouse since it is the background mouse strain used for all behavioural experiments in transgenic and knockout research et al. et al.et al. Prior to the main study, 12 mice were used to clarify the difference between continuous CORT exposure via the drinking water or by daily s.c. injection, the reversibility of physiological effects after treatment cessation and the reliability of oral glucose tolerance tests to indicate between-group changes in insulin sensitivity. No effect of 4 weeks CORT treatment via the drinking water or daily injection on glucose tolerance could be revealed. This is not in line with the severely compromised glucose tolerance found by Karatsoreos at al., Daily s.c. or i.p. injections of glucocorticoids solutions might be more precise and standardized with respect of the amount of drug delivered at a specific time point thereby avoiding fluctuations of circulating drug concentrations. However, the acute stress effect of the injections can easily and seriously confound the long-term effects of continuous high levels of glucocorticoids et al.In addition, the present study shows that CORT treatment significantly increased plasma insulin concentrations, whereas blood glucose levels remained unchanged. Insulin works as a key signal of blood glucose levels and mean plasma insulin levels can therefore be used to indicate the relative degree of insulin sensitivity with a higher plasma insulin concentration reflecting more insulin resistance Chronic stress-induced HPA-axis dysfunction is well known to increase the risk to developing major depression Long-term continuous treatment with exogenous CORT via the drinking water in the mouse seems a valid and reversible model of decreased insulin sensitivity mimicking both the physiological and the behavioural effects of hypercortisolaemia-induced IR which can ultimately develop in T2DM. The model therefore serves to further explore the causal direction and underlying mechanism of the strong association between IR and major depression, which up until today remains unresolved. As the model was established in C57BL/6NCrl mice, which is the genetic background mouse of many genetically manipulated mouse strains, including the APP/PS1 Alzheimer mouse, it additionally allows further investigation into the long-term effects of stress-induced IR and depression as risk factors for developing Alzheimer’s disease later in life.


There is strong evidence that post-exposure prophylaxis (PEP) with antiretroviral drugs in the timely management of occupational exposures sustained by healthcare workers decreases the risk of HIV infection and PEP is now widely used. Antiretroviral drugs have well documented toxicities and produce adverse events in patients living with HIV/AIDS. In the era of “highly active antiretroviral therapy”, non-adherence to treatment has been closely linked to the occurrence of adverse events in HIV patients and this ultimately influences treatment success but the influence of adverse events on adherence during PEP is less well studied.p < 0.05 was considered statistically significant.Following the introduction of a HIV post-exposure prophylaxis program in the Korle-Bu Teaching Hospital in January 2005, the incidence of adverse events and adherence were documented in occupationally-exposed healthcare workers (HCWs) and healthcare students (HCSs). Cohort event monitoring was used in following-up on exposed HCWs/HCSs for the two study outcomes; adverse events and adherence. All adverse events reported were grouped by MedDRA system organ classification and then by preferred term according to prophylaxis regimen. Adherence was determined by the completion of prophylaxis schedule. Cox proportional regression analysis was applied to determine the factors associated with the cohort study outcomes. Differences in frequencies were tested using the Chi square test and n = 28) in exposed HCWs/HCSs administered 3TC/AZT for 3 days, 91 % (n = 68) in exposed HCWs/HCSs administered 3TC/AZT for 28 days and 96 % (n = 50) in exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28 days. Nausea was the most commonly reported adverse events in all three regimens. Adherence was complete in all exposed HCWs/HCSs administered 3TC/AZT for 3days, 56 % (n = 42) in exposed HCWs/HCSs administered 3TC/AZT for 28 days and 62 % (n = 32) in exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28 days. In the Cox regression multi-variate analysis, exposed HCWs/HCSs administered 3TC/AZT for 3 days were 70 % less likely to report adverse events compared with exposed HCWs/HCSs administered 3TC/AZT for 28 days . Exposed HCWs/HCSs administered 3TC/AZT for 3 days were 75 % more likely to adhere to the schedule compared with exposed HCWs/HCSs administered 3TC/AZT for 28 days .A total of 228 exposed HCWs/HCSs were followed up during the study, made up of 101 exposed HCWs/HCSs administered lamivudine/zidovudine (3TC/AZT) for 3 days; 75 exposed HCWs/HCSs administered lamivudine/zidovudine (3TC/AZT) for 28 days; and 52 exposed HCWs/HCSs administered lamivudine/zidovudine/lopinavir-ritonavir (3TC/AZT/LPV-RTV) for 28 days. The frequency of adverse events was 28 %  and management of adverse events. Education on the need to complete PEP schedule  can lead to increased adherence, which is very critical in minimizing the risk of HIV sero-conversion. The present results also indicate that cohort event monitoring could be an effective pharmacovigilance tool in monitoring adverse events in exposed HCWs/HCSs on HIV post-exposure prophylaxis. There is evidence that post-exposure prophylaxis (PEP) with antiretroviral drugs in the timely management of occupational exposures sustained by healthcare workers (HCWs) decreases the risk of HIV infection and this type of approach is now widely used , 2. A caIn HIV-infected patients, combination regimens have proven to be superior to monotherapy regimens in reducing viral load , 6. ThusAntiretroviral drugs (ARVs) have well documented toxicities and produce adverse events in patients living with HIV/AIDS. These adverse events have been reported in several studies involving cohorts of patients with incidence rates as high as 54 % being reported in some instances , 10. In With the introduction of HAART, there have been considerable changes in the administered ARV cocktail which comes along with its own adverse events and adherence issues. The use of ARVs in PEP has shifted from the administration of single-drugs like zidovudine to multi-drug regimens involving dual or triple therapy with their associated issues of adverse events and adherence. Very few studies on adverse events and adherence in exposed HCWs/HCSs administered PEP has been conducted in developing countries, hence results from resource-limited settings like the Korle-Bu Teaching Hospital (KBTH) in Accra, Ghana is useful for an effective implementation and education of HCWs on PEP in these settings. Such studies also go a long way to aid program design and national policy in relation to PEP.The KBTH in Ghana with a workforce of about 1930 HCWs and 3330 healthcare students (HCSs), attends to over 3000 patients daily and also provides HAART services to over 20,000 HIV patients. In 2003, the National AIDS/STI Control Program (NACP) in Ghana provided guidelines on the provision of PEP in all sites that offer clinical care to patients living with HIV including KBTH, one of the four pilot sites for ART delivery at the time. KBTH has since then been offering HAART services to clients regularly.This study utilizes cohort event monitoring to follow exposed HCWs/HCSs for adverse events and adherence to PEP during a PEP service between January 2005 and December 2010. Cohort-event monitoring (CEM), a prospective and observational cohort study is an adaptation of prescription-event monitoring and it involves actively following up on patients within a defined time-frame for reports of adverse events . CEM empThis study was a prospective cohort analysis of HCWs/HCSs administered PEP at the KBTH in Accra, the premier tertiary referral hospital in Ghana, during the period of January 2005 to December 2010. A team of healthcare professionals made up of medical and pharmacy personnel were responsible for providing PEP service to exposed healthcare workers using an in-house risk assessment system previously described  as well The procedures and criteria for the administration of PEP and collection of data  have been described in a previous paper . ExposedActive follow-up for adverse events and adherence to prophylaxis schedule were performed by trained research assistants through telephone contact on days 3 and 10 after drug dispensing for those on the 3-day schedule and on days 3, 10, 20, 28 and 35 after drug dispensing for those on the 28 days schedule. In addition to the active follow-up, exposed HCWs/HCSs were asked to report events of medical concern  at any time during the follow-up period, noting especially the following signs; fever, rash, lymphadenopathy, dark-coloured urine, sore throat and bruising or bleeding from any part of the body. A structured questionnaire, interview guide was used to collect data from exposed HCWs/HCSs. Event data on adverse events was first recorded according to how the patient described the event (verbatim) and then reviewed qualitatively and coded using the Medical Dictionary for Regulatory Activities  terminology. All adverse events reported were grouped by MedDRA system organ classification (SOC) and then by preferred term (PT) according to prophylaxis regimen. It is important to note that unlike some CEM studies, the presence of adverse events prior to treatment initiation was not undertaken. All adverse events solicited post-treatment were new events that could conceivably be associated with PEP though no formal case-causality assessment was carried out on the individual reports.Adherence was determined by the completion of prophylaxis schedule assessed during the follow-up period by the use of a questionnaire. Non-adherence was defined as the inability to complete all the 3 days of the prophylaxis schedule for exposed HCWs/HCSs on the 3-day schedule and all the 28 days for exposed HCWs/HCSs on the 28-day schedule as prescribed.p < 0.10) were adjusted for in the multivariate analysis and reported as an adjusted hazard ratio. P < 0.05 was considered statistically significant.Each exposed HCW/HCS was given a unique study code which was used in data storage and management relating to that HCW/HCS. Data were double entered, cleaned and managed using Microsoft Access  and analysed using SPSS version 19 . Data were expressed as frequencies and percentages for categorical variables. Differences in frequencies and proportions were tested using the chi square test. The primary outcomes of interest were “adverse events” and “adherence”. Cox proportional regression analysis was applied to determine the factors associated with adverse events/adherence and reported as a crude hazard ratio in the univariate analysis. Variables significantly associated with adverse events or adherence in the univariate analysis  compared with those administered 3TC/AZT for 3 days, but not significantly different (p = 0. 832) from exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28 days. Over 80 % of the exposed HCWs/HCSs reported their exposure within 24 h and the median time between exposure and reporting was 2.0 h in both HCWs and HCSs. Follow-up for HIV testing was done at 6 weeks, 3 months and 6 months after exposure/HIV PEP administration and none of the HCWs/HCSs followed-up sero-converted.Results from Fig. n = 99) of exposed HCWs/HCSs administered 3TC/AZT for 3-days, 50 % (n = 41) of exposed HCWs/HCSs administered 3TC/AZT for 28 days and 53 % (n = 28) of those administered 3TC/AZT/LPV-RTV for 28 days. The 18–30 years age category represented the largest number of HCWs/HCSs administered all the three PEP regimens . Whilst risk assessment results for most of the exposed HCWs/HCSs administered 3TC/AZT for 3-days were low , the majority of exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28 days risk assessment results was high . The majority of exposed HCWs/HCSs administered 3TC/AZT for 28 days risk assessment results were either low risk  or medium risk . A vast majority of the reported exposures in general were percutaneous  with needle stick injuries being the most reported means of exposure .Table p < 0.001) in exposed HCWs/HCSs administered 3TC/AZT for 28 days  compared with exposed HCWs/HCSs administered 3TC/AZT for 3 days  but not significantly different (p = 0.236) from exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28 days .Out of a total of 228 HCWs/HCSs administered HIV PEP and followed up, 146 (64 %) reported at least one adverse event made up of three spontaneous reports and 143 reports from active surveillance. The proportion of those who reported at least one adverse event was significantly higher (p < 0.001) (Table p = 0.817) in the reporting of adverse events between exposed HCWs/HCSs administered 3TC/AZT for 28 days and exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28 days in the multivariate analysis.Table 1) Table . There wThe most frequently observed type of adverse event reported in all the three PEP regimens was gastrointestinal in nature Table . Nausea,There were three spontaneous reports of adverse events which ended up in hospitalisation but all recovered. The first was an exposed female HCW of 26 years administered 3TC/AZT/LPV-RTV for 28 days who reported of dizziness, excessive flatulence, headache, nausea, vomiting, restlessness and dark pigmentation of finger & toe nails (which later came off) and was hospitalised for three days. The second case which resulted in hospitalisation for seven days involved a 29-year old exposed pregnant female HCW (in the first trimester of pregnancy) who reported of abdominal pains, profuse diarrhoea, fatigue, nausea and bleeding which later resulted in spontaneous abortion. The third case involved an exposed female HCW of 42 years who was administered 3TC/AZT for 28 days and reported of fatigue, headache and weakness which according to her resulted in a “near death” experience. She was hospitalised for three days. These three serious adverse events all involving females have been reported to the National Pharmacovigilance Centre at Ghana’s Food and Drugs Authority.n = 175) in this study. All the 53 PEP exposed HCWs/HCSs who defaulted in their 28-days prophylaxis schedule did so within 14 days of medication initiation. None of the exposed HCWs/HCSs on the 3-day schedule defaulted in medication adherence. Comparing the different PEP regimens administered, adherence to the prophylaxis schedule was significantly higher (p < 0.001) in exposed HCWs/HCSs administered 3TC/AZT for 3-days  compared with exposed HCWs/HCSs administered 3TC/AZT for 28-days  and exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28-days . However, there was no statistical difference (p = 0. 534) in adherence proportion between exposed HCWs/HCSs administered 3TC/AZT for 28-days and exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28-days.Complete adherence to PEP schedule was 77 % (p = 0.008). There was no statistical significant difference in terms of adherence between exposed HCWs/HCSs administered 3TC/AZT for 28 days and exposed HCWs/HCSs administered 3TC/AZT/LPV-RTV for 28 days (p = 0.660).Table p = 0.003). All the 53 exposed HCWs/HCSs who did not adhere completely to their PEP medication schedule cited adverse events as their reason for non-adherence.In a separate analysis to determine the association between the two study outcomes (report of adverse events and adherence), exposed HCWs/HCSs who never reported of adverse events were 57 % more likely to adhere to their medication schedule compared with exposed HCWs/HCSs who reported of at least one adverse event  in exposed HCWs/HCSs undergoing different regimens, more so with the 28-day regimens. Again, it showed that serious adverse events leading to hospitalization were common, occurring in this case at a frequency higher than 1 in 100 (3 serious adverse events in 228 exposed HCWs/HCSs). All the exposed HCWs/HCSs who reported serious adverse events were females and were on 28 days regimen schedule. The importance of the duration of treatment on adverse events and adherence is starkly demonstrated by the fact that adverse events were low and adherence excellent in the 3 days regimen schedule whilst adverse events were more common and adherence varied between 56 and 62 % in the 28 days regimen schedule.In this study exposed HCWs/HCSs administered PEP were allowed to describe their complaints in their own context before the use of the system organ class (SOC) classification to group the adverse events reported. In general, these exposed HCWs/HCSs administered PEP were not on other medications of note and therefore related these events to the anti-retroviral medications administered. The events reported did not exist prior to initiation of therapy. The present results indicate an overall adverse event frequency of 64 %, which is lower than the results of other studies where the frequency of adverse events was reported to be between 70 and 76 % , 18, 26.As expected, the adverse events reported were consistent with the product profiles (Summary of Product Characteristics-SmPC) of the PEP medications in use. The most frequently cited symptoms were gastrointestinal in nature; vomiting, diarrhoea, and nausea followed by neurological symptoms like headache, weakness and fatigue which is similar to results from other PEP studies , 19, 28.The study recorded three serious adverse events – all cases of hospitalisations following initiation of PEP. Worth noting was the case of dark pigmentation (hyper pigmentation) of finger and toe nails reported by a patient on 3TC/AZT/LPV-RTV for 28 days which incidentally has been reported in some other studies as a cutaneous adverse reaction of zidovudine (AZT), mostly in patients on chronic treatment of HIV [This study could not establish any association between the report of at least one adverse events and factors such as gender, age, profession, outcome of risk assessment and type of exposure in the multivariate analysis although other studies have associated adverse events with gender . The preResults from this study indicate an overall adherence rate of 77 %. Although this adherence rate is far higher than reported in some studies , 26, it The use of the CEM technique in this study has led to the successful monitoring of adverse events and adherence to PEP schedule. This study has demonstrated that is it possible to effectively conduct an active safety monitoring of ARVs used in PEP in resource limited settings like the KBTH in Accra, Ghana. The safety data obtained from this CEM study are consistent with the safety profile expected for the medicines concerned. Since the tendency to passively/spontaneously report adverse events and adherence in HIV negative exposed HCWs/HCSs on PEP is generally low, CEM will become a key pharmacovigilance method in resource-limited settings due to its ability to aid in rapid identification of signals and to effectively compare regimen and the effect of treatment type and duration on both adverse events and adherence.Most of the exposed HCWs/HCSs were willing to be followed up by phone and loss to follow up was reasonably low compared to other studies in these settings. Although the inability to follow exposed HCWs/HCSs administered PEP beyond 6 months to monitor possible long term toxicities is a limitation, monitoring of long term toxicities was not the focus of the current study. Since laboratory tests for liver enzymes, full blood count, etc. were not conducted, it is not known to what extent the medicines might have caused abnormalities which could only be detected via laboratory testing. However the relatively shorter period of 28 days given for PEP does not permit extrapolation of data to the identification of possible long-term toxicities that might have occurred if these products had been taken for long periods. The results cannot therefore be extrapolated to verify or refute known HAART induced toxicities like hepatitis or zidovudine-induced anaemia  which ocAlthough the results of this study cannot be necessarily generalized across HIV/AIDS treatment sites in Ghana or in West Africa and other resource-limited settings, the incidence of adverse events and non-adherence provides an insight into what to expect in such settings and this information could be used for patient education and the education of HCWs as PEP provision is scaled up. This study has shown the importance of focused cohort studies in generating important safety information in resource-limited settings where the spontaneous reporting ADR systems yield very little data and where healthcare systems e.g. absence of electronic health records and weak laboratory infrastructure, do not permit collection of safety data in any systematic manner. A possible limitation of the current study is the in-house medication adherence metrics used. Although medication adherence metrics are available including the one validated by Morisky et al. , none waIn conclusion, it is possible to use active follow-up method via mobile phone calls to monitor both adverse events and adherence to prophylaxis regimen in HIV-negative PEP subjects. The study showed that adverse events to PEP are very common and could be severe or serious requiring hospitalization for management in some cases and that the nature and frequency of adverse events collected in the current study were consistent with available information on the use of those antiretroviral drugs. Much emphasis should be attached to follow-ups in order to advise and act promptly when issues arise. This study also indicated the need for education of exposed HCWs/HCSs on the importance of complete adherence to HIV PEP in order to minimize the risk of HIV sero-conversion.


This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many  Here we utilize Nanostring digital assays and show their utility for estimating copy number of 186 multicopy genes and tandem repeats. By analyzing patterns of single nucleotide variation around these variants, we show that copy number variation at the vast majority of tandem repeat variations is not effectively tagged by nearby SNPs, and thus standard genome-wide association studies that focus on SNPs provide little or no information about such variants. By comparing patterns of tandem repeat copy number with variation in local gene expression and DNA methylation, we also identify extensive functional effects on local genome function. This includes an example of a non-coding macrosatellite repeat, expansion of which exerts a repressive effect on a nearby gene accompanied by accumulations of local DNA methylation. Finally, comparison of diverse human populations with a number of primate genomes shows that many of these sequences have undergone extreme changes in copy number during recent human and primate evolution, and show signatures that suggest possible selective effects. Overall, we conclude that multicopy genes and macrosatellites represent a highly variable fraction of the genome with important functional effects that has been systematically ignored by previous studies. More than half of the human genome is composed of various types of repetitive elements −4 for dinucleotide repeats, and 8.5–15×10−4 for tetranucleotide repeats −8de novo mutations per site per meiosis Despite representing a significant source of genomic variation CCCCGCCCCGCG expansions upstream CSTB [OMIM# 601145], Huntington disease caused by a CAG expansion within the coding region of HTT [OMIM# 143100], and Fragile X syndrome caused by a CGG expansion in the 5′UTR of FMR1 [OMIM# 300624]. In addition to causing disease, changes in the length of microsatellites can exert effects on gene function and quantitative traits While microsatellite repeats have found common usage in linkage studies and forensics, several human disorders are known to be caused by extreme expansions of tandem repeats located within or near protein-coding genes β-defensin genes at 8q23 is associated with susceptibility to psoriasis In contrast to microsatellites, macrosatellites and multicopy genes are less well studied. Although the functions of most multicopy genes located in tandem arrays are poorly characterized, recent studies have begun to associate copy number variation (CNV) of several of these with human traits. For example, CNV of the antimicrobial FMR1 via hypermethylation DUX4 gene, in combination with a permissive haplotype background, underlie facioscapulohumeral muscular dystrophy (FSHD) In some cases, CNV of tandem repeat arrays has also been associated with epigenetic effects. For example, the promoter expansions of the CGG repeat that causes Fragile X are associated with silencing of In this study we have adapted Nanostring technology, a multiplexed digital counting method based on fluorescent barcodes initially developed for gene expression analysis https://usegalaxy.org/), using the complete list of RefSeq genes as the two input files, with gene name as the joined field. We filtered the output to only retain genes that had >1 copy at a non-overlapping position on the same chromosome or chr_random, identifying 180 putative multicopy genes in the hg18 reference assembly. We designed a custom Nanostring probe set targeting 173 of these genes, in addition to 13 non-coding macrosatellites and 2 intragenic coding repeats  of our probes have one or more BLAT alignments of ≥95% identity on a chr_random, and 34% (60 of 175 genic probes) lie within 50 kb of an assembly gap, representing a >27-fold enrichment compared with all RefSeq genes. In total, 86 of the 337 (26%) euchromatic assembly gaps in the hg18 assembly lay within 50 kb of a multicopy gene, suggesting that the highly repetitive and polymorphic nature of multicopy genes are a significant source of genome assembly errors. As a result, the true copy number of such regions is often highly under-represented in the genome assembly. While the mean number of BLAT alignments of our probes in the haploid hg18 was 5.4 (median = 3), the estimated mean diploid copy number of these loci in the 165 HapMap individuals was 22 (median = 21.5).CT47, TBC1D3, PRR20A, REXO1L1, and CCL3/CCL4). In each case, these independent replicates yielded highly concordant results (mean R2 = 0.88) (CT47 tandem array by pulsed-field gel electrophoresis Southern blots REXO1L12 = 0.92). We also compared Nanostring counts for the 8p23.1 β-defensin gene cluster with copy number measurements for this locus made using the paralog ratio test (PRT) 2 = 0.90, To assess the reproducibility of Nanostring assays we first compared data generated from two independent probes, each of which target different parts of five genes that show highly variable copy numbers ( = 0.88) , indicatWhile Nanostring assays provide counts that are proportional to underlying copy number, relative counts given by different assays can vary depending on individual probe efficiencies. To derive absolute copy numbers in each individual, we therefore compared Nanostring probe counts with corresponding copy number estimates from whole-genome shotgun read depth analysis from the 1000 Genomes Project DUX4, NBPF10, USP17 and REXO1L1) showed mean diploid copy numbers >100 within the normal human population, and these loci also showed the highest variability  loci tested showed copy number variation among the 165 HapMap individuals. Several of these loci (iability . For exaST (VST) values for each probe in our design. Elevated (>0.2) values of VST indicate sites with highly divergent copy numbers in different populations, which can be a signature of positive selection ST values >0.2 among European, African and Asian populations . To quantify this inter-population variance we calculated ANOVA-Fulations , indicatWe performed an intersection of our probe locations with a list of 8,598 CNVs detected by a previous array CGH study that utilized ∼21 million probes spaced throughout the human genome 2>0.8 2 = 0.22 between the top ranked SNP and probe count. Only four loci (3.4% of the loci tested) showed an R2≥0.8 with any SNP in at least one of the three populations studied . We obtained very similar results when using published gene expression data generated using RNAseq (data not shown) To measure the functional effects of changes in copy number of tandemly repeated sequences we performed correlations with local gene expression levels in human lymphoblastoid cell lines. We used published gene expression data for 58 CEU and 59 YRI HapMap individuals y number . SomewhaZFP37 gene. In contrast, the strongest negative correlations were observed for multiple CpGs around the CCL3/CCL4 locus , a 5.4 kb a GC-rich repeat (60% G+C) which lies 2 kb proximal and varies between 4–43 diploid copies in human  with copy number of the adjacent MSat12, which lies 24 kb distal. This correlation of MSat12 copy number with ZNF558 expression was detected independently in both European and African populations, showing that these effects are reproducible and not specific to single ethnic groups  to non-synonymous (dN) amino-acid replacement between humans and other primates  as an additional measure of evolutionary selection. Using a curated set of human:primate orthologs Utilizing a novel multiplexed digital genotyping technology, we have performed a detailed analysis of copy number variation of multicopy genes and tandemly repeated macrosatellites in the human genome. We identify many interesting biological insights at these loci. This includes (i) extremely high levels of CNV among both different human populations and primate species; (ii) an almost universal lack of LD between CNV of these regions and nearby SNP markers, meaning that CNV of the vast majority of multicopy genes and macrosatellites is not tagged by flanking SNP markers, and that effective interrogation of these loci requires specific methods capable of effectively assaying these regions; (iii) significant associations between tandem repeat copy number and local gene expression and epigenetics; and (iv) signatures suggestive of positive selection acting at many of these loci. Thus, despite being largely ignored by most genome-wide studies, this class of genomic variation shows many features that suggest important functional effects.2 = 0.76), together with a weaker but significant inverse correlation between MSat10 copy number and expression of the adjacent gene ZFP37, which lies 2 kb proximal to the repeat. ChIP-seq analysis also showed the presence of heterochromatic marks associated with MSat10 which extend proximally to ZFP37. Thus our data suggest heterochromatinization of the MSat10 locus at high repeat copy number with a resulting suppression of local gene expression. A similar inverse relationship was also observed between MSat12 copy number in 19p13.2 and expression of the adjacent gene ZNF558, which lies 24 kb distal to the repeat, although we did not detect any significant inter-individual variation of DNA methylation at this locus. Based on transgenic studies, it has been proposed that repeat induced gene silencing represents a system for the protection of eukaryotic genomes against hostile sequence elements that can integrate into the genome in high copy numbers, such as viruses and transposons ZFP37 and ZNF558 lie just 2 kb and 24 kb from the macrosatellites whose copy number they are inversely correlated with, respectively.Of particular note, our analysis identifies an example consistent with repeat induced gene silencing operating in normal human genomes associated with increased copy number of MSat10, a GC-rich 5.4 kb tandemly repeated motif located in 9q32 . We observed a strong positive correlation between copy number of MSat10 and local DNA methylation level , and 13 non-coding macrosatellite repeats identified in a previous study 180 multicopy genes were identified by performing a self-join operation using RefSeq to identify genes that had >1 copy at a non-overlapping position on the same chromosome or chr_random. Custom Nanostring probes were successfully designed for 173 of these genes, in addition to two intragenic tandem repeats  for which we designed two independent probes targeting different parts of the gene to measure assay reproducibility. This probe set was used to genotype 165 unrelated Phase I HapMap individuals , and five species of primate (one Pan troglodytes (chimpanzee), one Pan paniscus (Bonobo), two Gorilla gorilla (gorilla) individuals, one Hylobates sp. (gibbon), and one Macaca arctoides (Stump-tailed macaque)) ) .Pan troglodytes (panTro2), Gorilla gorilla (Gor3.61) and Macaca mulatta (rheMac2). BLAT results ≥90% identity, allowing for 2 bp of insertion/deletion, are shown in Each probe sequence was queried against the hg18 human reference genome using BLAT. Alignments of ≥90% identity, allowing for 1 bp of insertion/deletion, are shown in SRY and invariant X chromosome loci were included as both gender controls and to provide internal performance control data between males and females. Two probes (SCXB and TAF9B) yielded mean counts <100 across all HapMap individuals and were excluded from further analysis. Background subtraction was first performed on raw counts by subtracting the mean count of the eight negative control probes in each sample from the raw count of all test probes. The ten invariant autosomal probes were then used to derive a normalization factor for each sample to account for technical variables such as varying amounts of input DNA or hybridization efficiency between assays. The normalization factor per sample was derived by calculating the mean count of the ten invariant control probes in that individual divided by the mean count for all normalization probes across all samples. Raw counts for each of the 195 probes in each individual where then multiplied by the normalization factor for that sample to produce normalized probe counts, which were used in all downstream analysis. We defined copy number variable probes as those with either a coefficient of variation (CV) ≥0.1 among the 165 HapMap samples, or those that had counts ≥30% higher or lower than the population mean in ≥2 individuals.The probe set included eight negative control probes that target artificial sequences, and ten normalization probes that target autosomal loci that are invariant in copy number. Additional probes for Each Nanostring probe provides a count that is directly proportional to copy number of the target sequence in that individual. However, the relative counts given by different probes can vary depending on individual probe efficiencies. We therefore converted each probe count to absolute copy number using an independently calibrated data set. To derive absolute copy numbers in each individual, we compared our probe data with estimated copy number from whole-genome shotgun read depth analysis of individuals tested as part of the 1000 Genomes Project http://hapmap.ncbi.nlm.nih.gov), and SNPs with minor allele frequency (MAF) <0.1, Hardy Weinberg Equilibrium (HWE) p<0.05, or tri-allelic states were removed. To calculate LD between copy number for each of the 116 variable Nanostring probes and local SNPs, SNP genotypes were converted to integers  and Pearson correlations with normalized probe counts performed to derive the coefficient of determination (R2). For each Nanostring probe, we extracted SNP genotype data within ±250 kb of each BLAT match of the probe sequence with ≥95% identity. Significance testing was performed using a permutation test in which the SNP genotypes and probe counts were randomly permuted across the samples in each dataset 10,000 times. For loci on the X and Y chromosomes correlations were calculated separately using male and female individuals. A median of 227 SNPs were tested per probe (range 8–1692) (To assess the degree of linkage disequilibrium (LD) between copy number of loci targeted by each probe and flanking SNP markers, we performed correlation analyses between probe counts and SNP genotypes. HapMap Phase II SNP genotypes for the 165 unrelated individuals studied  were downloaded  . As SNPs 8–1692) .2 expression <6, or interquartile range <0.25 were removed. A median of 19 transcripts were tested for association with each probe (range 1–306). Pearson correlations were performed to derive the correlation coefficient (R) and coefficient of determination (R2) for each transcript:probe pair. Correlations for loci on the X and Y chromosomes were performed separately for males and females. Significance testing was performed by permutation in which the gene expression data and Nanostring probe counts were permuted across the samples in each dataset 10,000 times.We performed correlation analysis of Nanostring probe counts with steady-state mRNA levels of nearby transcripts. We used published Affymetrix exon array data available for 58 CEU and 59 YRI samples in our cohort We used published methylation data S8). In order to avoid the confounder of X chromosome inactivation, we calculated correlations on chromosome X using male samples only using the combined CEU and YRI individuals in order to ensure sufficient sample size for a meaningful analysis , resulting in a total of 98,941 methylation probes for which we measured the effect of copy number variation on the methylation patterns. We calculated Spearman's correlation (R) between Nanostring probe count and methylation levels , considering CEU and YRI populations as a single group as well as in either population separately . In the analysis .ST statistics (ANOVA-FST). Briefly, for each Nanostring probe we performed ANOVA across the European (CEU), African (YRI) and Asian (CHB) populations, and extracted the variance components from the ANOVA analysis, representing the between- and within-group variances. Variances were normalized within each population based on the total number of samples for that population. For loci on the X and Y chromosomes ANOVA-FST was calculated separately for female  and male  individuals, with the final FST reported being the mean of the FST male and FST female.We identified sites showing evidence of population stratification among the three HapMap populations tested using ANOVA-based FTo measure the extent of selective pressure on multicopy genes tested in our analysis during primate evolution, we analyzed the dN/dS ratios (ratio of the number of non-synonymous substitutions to the number of synonymous substitutions) of these genes in human versus chimpanzee, orangutan and macaque. High confidence one-to-one orthologs for all human Refseq genes and their dN/dS ratios were extracted from Ensembl v51 Data for the five primate species tested were normalized and background corrected using the same method as in human. However, because probes were designed against the human reference sequence which is potentially divergent from the primate genomes, we first filtered the primate data to remove low-confidence data points that likely resulted from sequence divergence between human probe and primate target sequence. To avoid normalization artifacts, data normalization in each primate species was performed using only those invariant control probes whose count differed <20% from the relative mean signal for that probe in the 165 HapMap individuals . To ensure robust measurement of the relative copy number of each locus in primate versus human that was not influenced artifactually by poor probe binding, we retained data only for those probes exceeding a minimum identity threshold in each primate species. For loci showing a relative increase in copy number in a particular primate species versus human, we retained only those data points for which the probes had ≥95% sequence identity in that species. However, as mismatches between probe and target will generally result in reduced binding efficiency, we utilized a more stringent threshold of ≥98% sequence identity for any probes that showed a relative decrease in copy number in primate versus human. For CNVs on the sex chromosomes, relative copy number change in human versus primate was only based on individuals of the same gender.in vitro using T7 polymerase, T-cleaved and processed using MALDI-TOF MS. DNA methylation levels were quantified using the EpiTYPER software.DNA methylation levels were analyzed by MassARRAY EpiTYPER (Sequenom Inc.), using base-specific cleavage and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Briefly, DNA samples isolated from HapMap lymphoblastoid cell lines  were bisulfite converted using the EpiTect Bisulfite Kit (Qiagen) and amplified by PCR using promoter-tagged reverse primers and 10-mer sequence-tagged forward primers specific to each region . AmplifiHaeIII (New England Biolabs) to fragment the repeats. 20 ng of digested DNA was combined with QuantaLife Master Mix, PCR primers and VIC and FAM MGB labeled probes . DNA from MS4221 and from 3 HapMap individuals with known MSat10 copy number were digested overnight at 37°C with d probes . DropletChIP was performed on lymphoblastoid cell line MS4221 Figure S1TBC1D3, REXO1L1, PRR20A and CCL3/CCL4 all show high concordance, indicating that Nanostring probe counts provide reproducible measurements that are proportional to copy number over a wide dynamic range.Independent Nanostring assays targeting polymorphic high copy number genes yield concordant results. Measurement of gene copy number using two independent probes targeted to different parts of the genes (TIF)Click here for additional data file.Figure S2REXO1L1 tandem array by pulsed-field gel electrophoresis Southern blots show excellent correlations with Nanostring counts. (b) Comparison of Nanostring counts for the 8p23.1 β-defensin gene cluster with copy number measurements for this locus made using the paralog ratio test (PRT).Comparison of copy numbers generated using Nanostring technology against other technologies for measuring high-copy number sequences. (a) Copy number estimates based on direct fragment sizing of both the (TIF)Click here for additional data file.Figure S3Comparison of copy numbers generated using Nanostring technology against those estimated by read depth analysis from whole genome shotgun sequencing. Each scatter plot shows normalized Nanostring counts for one probe versus copy number estimates of the same gene or macrosatellite locus made in the same individual by read depth analysis (TIF)Click here for additional data file.Figure S4PRAMEF14 shows the most extreme change detected, with an ∼18-fold increase in copy number in chimpanzee compared to the human mean, suggesting >200 copies of this gene in the chimpanzee individual tested. Note that as many probes do not have a perfect match in one or more of the other species tested, particularly in the more divergent species such as Gorilla, Gibbon and Macaque, many of these copy number expansions observed in different primates are almost certainly under-estimates.Loci showing highly divergent copy number between humans and other species of primate. 20 multicopy genes and macrosatellites showed ≥3-fold gain in copy number in at least one primate species versus the human mean measured by Nanostring assays. Each plot shows the relative fold-change versus the human mean. (TIF)Click here for additional data file.Figure S5green) compared to all RefSeq genes (red) for human versus Orangutan and Macaque. There is a significant enrichment for elevated rates of non-synonymous substitution in multicopy genes versus the genome average in both species. This excess of non-synonymous amino-acid changes in recent primate evolution at multicopy genes is consistent with either reduced selective constraint and/or selection for proteins with altered function. Similar results are obtained when comparing human with chimpanzee Click here for additional data file.Table S1Description of Nanostring probe sequences used in this study and their mapping coordinates in hg18.(XLSX)Click here for additional data file.Table S2Nanostring counts for each probe in the 165 Hapmap individuals. Nanostring probe counts represent processed data after normalization and background correction of raw counts See . Abbrevi(XLSX)Click here for additional data file.Table S3Summarized absolute copy numbers of multicopy genes and macrosatellites in 165 HapMap individuals. Abbreviations: INV: Invariant, VAR: Variant.(XLSX)Click here for additional data file.Table S42 value with probe count within ±250 kb. Abbreviations: CEU, CEPH Europeans from Utah; YRI, Yoruba from Ibadan Nigeria; CHB, Han Chinese in Beijing, ALL: Includes CEU, YRI, CHB individuals combined.(XLSX)Click here for additional data file.Table S52 value with probe count within ±250 kb after removing SNPs that: (i) overlap regions of common copy number variation Click here for additional data file.Table S6CCR5, FCGR, GSTM, GSTT2, KRTAP1-1, MAFG, MAGEA9, UGT2B15) shows significant correlation (p<0.01) with their own expression level. Abbreviations: CEU, CEPH Europeans from Utah; YRI, Yoruba from Ibadan Nigeria, All: CEU, YRI and CHB individuals combined; R and R2 are derived from Pearson correlations.(XLSX)Click here for additional data file.Table S7(XLSX)Click here for additional data file.Table S8Filtered results of correlation analysis between probe counts and methylation level of autosomal CpGs within ±500 kb of each probe BLAT position. Reported in this table are pairwise correlations between Nanostring counts and CpG methylation levels  that show permutation p<0.01 in the combined CEU and YRI populations and which also validate (unadjusted p<0.01) in each in each population separately . In addition the effect size on methylation, as measured by the absolute normalized slope, must be >0.1. Abbreviations: CEU, CEPH Europeans from Utah; YRI, Yoruba from Ibadan Nigeria.(XLSX)Click here for additional data file.Table S9Filtered results of correlation analysis between probe counts and methylation level of X-linked CpGs within ±500 kb of each probe BLAT position. Reported in this table are pairwise correlations between Nanostring counts and CpG methylation levels  that show permutation p<0.01 in the combined CEU and YRI male populations (chromosome X probes only). In addition the effect size on methylation, as measured by the absolute normalized slope, must be >0.1. Abbreviations: CEU, CEPH Europeans from Utah; YRI, Yoruba from Ibadan Nigeria.(XLSX)Click here for additional data file.Table S10Probe alignments, probe counts and relative copy numbers obtained in five species of primate compared to the human mean.(XLSX)Click here for additional data file.Table S11Description of primers used for Sequenom MassARRAY EpiTYPER and QuantaLife droplet digital PCR.(XLSX)Click here for additional data file.