SUMO modification of BLM controls the switch between BLM's pro- and anti-recombinogenic roles in homologous recombination following DNA damage during replication. BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased γ-H2AX foci. Because the increased γ-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess γ-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.The gene mutated in Bloom's syndrome,  Replication is the process in which cellular DNA is duplicated. DNA damage incurred during replication is detrimental to the cell. Homologous recombination, in which DNA sequences are exchanged between two similar or identical strands of DNA, plays a pivotal role in correcting replication processes that have failed due to DNA breakage and is tightly regulated, because deficient or excess recombination results in genomic instability. Previous studies have implicated the DNA-processing enzyme BLM in the regulation of homologous recombination; BLM is defective in Bloom's syndrome, which is characterized by excess recombination and cancer susceptibility. Here, we show that modification of BLM by the small protein SUMO controls BLM's function in regulating homologous recombination at sites where DNA replication failed. We showed that cells expressing a SUMO-deficient mutant of BLM accumulated more DNA damage and displayed defects in repair by homologous recombination. An enzyme involved in homologous recombination, RAD51, displayed a defect in localization to sites where DNA replication failed. Our data support a model in which SUMO modification regulates BLM's function in homologous recombination by controlling the localization of RAD51 to failed replication sites. BLM gene BLM gene encodes a DNA helicase of the RecQ family, which is an evolutionarily conserved group of enzymes that operates at the interface of DNA replication, HR, and DNA repair Homologous recombination (HR) is a high-fidelity DNA repair mechanism that functions to rejoin double-strand breaks (DSBs) and restart broken replication forks. A major outcome of the repair of replication fork damage by HR is the generation of sister-chromatid exchanges (SCEs), which result from resolution of Holliday junctions during HR repair The RecQ helicases are DNA-dependent ATPases that can translocate on single-stranded DNA (ssDNA) with 3′ to 5′ directionality Saccharomyces cerevisiae, the polymerase processivity factor PCNA (proliferating cell nuclear antigen) is SUMOylated, and PCNA SUMOylation recruits the DNA helicase Srs2 to the fork, which functions to prevent aberrant recombination events between sister chromatids MMS21 accumulate RAD51-dependent cruciform structures at damaged replication forks sgs1 deletion mutants Modification by the small ubiquitin-related modifier (SUMO) has emerged as an important regulator of HR We have previously shown that BLM is SUMOylated and that failure to SUMOylate BLM results in changes in BLM's nuclear distribution In the present study, we aimed to determine how SUMOylation regulates BLM's function in maintaining genomic integrity. We hypothesized that cells that express SUMO-mutant BLM have a DNA repair defect. Characterization of cells that expressed SUMO-mutant BLM revealed that SUMOylation of BLM regulates its association with RAD51 and its function in HR-mediated repair of damaged replication forks. Our data support a model in which SUMOylation of BLM acts as a switch to regulate its effects on recombination.Because BLM functions at damaged replication forks and γ-H2AX is a marker for DNA damage, we hypothesized that SUMO-mutant BLM is defective in repair of damaged replication forks. To gain insight into this question, we introduced GFP-BLM expression constructs into the BS cell line GM08505, isolated clones that stably expressed either normal BLM (BLM+ cells) or SUMO-mutant BLM (SM-BLM cells), and studied these clones for responses to replication fork damage. We treated SM-BLM and BLM+ cells with 0.5 mM hydroxyurea (HU) for 24 h, which stalls replication forks, and quantified the production of γ-H2AX by immunofluorescence, Western blot, and flow cytometry analyses . The 24-As we reported previously, untreated SM-BLM cells exhibited higher levels of γ-H2AX foci compared to untreated BLM+ cells  . In the We also analyzed the numbers of DSBs in cells treated with camptothecin (CPT), which generates replication-associated DSBs p = 0.01), demonstrating that SM-BLM cells have increased sensitivity to HU treatment alone. Similarly, a 24-h treatment with etoposide induced an 11.3% increase in cell death in BLM+ cells compared to a 20.7% increase in SM-BLM cells (p = 0.003), demonstrating that SM-BLM cells are also hypersensitive to etoposide treatment compared to BLM+ cells. After HU pretreatment, etoposide induced a 13.5% increase in cell death in BLM+ cells, which was similar to the level of cell death observed without HU pretreatment (11.3%) (p = 0.84), whereas after HU pretreatment, etoposide induced a 43.1% increase in cell death in SM-BLM cells, which was 2-fold greater compared to the level observed without HU pretreatment (20.7%) (p<0.001). As expected, BS cells that lack BLM protein are also hypersensitive to DNA damage encountered during S phase. In corroboration of these results, in colony survival assays, we also observed increased sensitivity of SM-BLM cells to CPT compared to BLM+ cells , HU treatment had almost no effect on the levels of SCEs in SM-BLM cells . The numbers of HU-induced SCEs in BLM+ compared to SM-BLM cells was significantly different (p<0.001). In contrast to SM-BLM cells, HU induced a large increase in SCEs in BS cells . Whereas BLM+ cells exhibited a 2.3-fold increase in RAD51 foci from 14.4 to 33.6 foci/cell in PCNA-positive cells, SM-BLM cells exhibited no increase in total RAD51 foci from 19.7 to 21.0 foci/cell . SynchroAltogether, these data demonstrated that there is a dramatic defect in RAD51's recruitment to and/or retention in repair foci induced by replication stalling. The RAD51 localization defect could explain both the impairment of HR after replication stalling and the excess DSBs observed in SM-BLM cells.To investigate the mechanism that might explain the RAD51 localization defect, we considered the possibility that RAD51 interacts with SUMO noncovalently, which would facilitate interaction between RAD51 and SUMOylated BLM. To test this hypothesis, we assayed for possible noncovalent interactions between RAD51 and SUMO and also for possible effects of covalent SUMOylation of BLM on its interactions with RAD51 . In an iAltogether, these data demonstrated that RAD51 contains a SUMO binding site(s) and that SUMOylation of BLM can affect its interactions with RAD51. The data support the hypothesis that SUMOylation of BLM facilitates repair of damaged replication forks by HR by modulating RAD51's recruitment and/or retention at repair sites.The data presented here demonstrate the importance of BLM SUMOylation in the repair of damaged replication forks. Failure to SUMOylate BLM resulted in excess damage-induced repair foci, DSBs, and hypersensitivity to DNA damage. Importantly, in SM-BLM cells, replication stalling by HU did not stimulate HR as measured by SCEs, suggesting a defect in RAD51 function. Consistent with these data, RAD51 failed to accumulate at stalled forks. SUMO-mutant BLM exerts a dominant effect, because excess γ-H2AX foci were induced by expression of SUMO-mutant BLM in HeLa cells, which express endogenous normal BLM The steady-state levels of DSBs in cells are a function of the rate at which DNA damage accumulates and the rate at which it is repaired. We observed that SM-BLM cells exhibited greater numbers of DSBs than BLM+ cells under a variety of conditions . For exaRecent work has shown that BLM is present on a class of ultrafine anaphase bridges Alternatively, BLM SUMOylation could promote HR-mediated repair of broken forks through the recruitment and/or retention of RAD51 at damaged forks. RAD51 is the DNA recombinase essential for HR-mediated DNA repair, and previous studies have demonstrated that RAD51 and BLM interact specifically in DNA-damaged cells BLM null cells Escherichia coli RecQ helicase Previous cell and biochemical studies have led to the view that BLM has both pro- and anti-recombinogenic functions. Most notably, BLM is important in stabilizing damaged replication forks SUMOylation of BLM could regulate the recruitment and/or the retention of RAD51 at sites of DNA damage through several different mechanisms. SUMOylation could dissociate BLM from broken DNA ends, where it might otherwise inhibit RAD51 binding and function by displacing RAD51 from ssDNA or by unwinding D-loops SUMOylation of BLM is likely to have multiple roles. In addition to regulating its activity at sites of DNA damage as revealed in the current study, BLM SUMOylation may also be important in mediating the localization of BLM to PML-NBs in undamaged cells. SUMO modification can function to retain proteins in the PML-NBs sgs1 mutants and mutants of the SUMO-specific E3 ligase gene mms21 accumulate aberrant cruciform structures at damaged replication forks Our results have broad implications for understanding not only how the integrity of replication forks are maintained under stress but also how SUMO modification regulates its substrates, because many proteins in the DNA repair and signaling pathways are SUMO substrates. Although the role of SUMO in HR function is not yet understood, it is clear that SUMOylation plays multiple roles in regulating the HR pathway through modifications of various HR factors, including Sgs1 For BLM Western analysis, rabbit polyclonal anti-BLM antibodies raised against the first 431 amino acids of human BLM The full-length BLM cDNA was cloned into the EGFP-C1 vector (Clontech), which produced a GFP-BLM fusion protein with GFP at the N-terminus of BLM, as described previously http://facs.scripps.edu) software. To examine the percentage of cells in S phase, cells were treated with HU, released, and then analyzed at times after release using the BrdU Flow Kit (BD Biosciences) according to the manufacturer's instructions. BLM+ and SM-BLM cell clones in the logarithmic phase of cell proliferation had comparable proliferation rates, with cell-doubling times equal to ∼30 h for each clone examined.To measure DNA content and percentage of cells in each phase of the cell cycle, cells were harvested by trypsinization and fixed in 70% ethanol for >3 h at −20°C. After fixation, cells were pelleted and resuspended in a solution of 1× phosphate buffered saline  containing propidium iodide (10 µg/ml) and RNase A (0.1 mg/ml). The fluorescence intensities of the propidium iodide–stained cells were measured using a FACScalibur (Becton-Dickinson), and data were analyzed with CellQuest (Becton-Dickinson) and WinMDI . Fixation and staining was performed as described previously 2) in at least one Z-stack in which the fluorescence intensity was greater than the background fluorescence intensity of the nucleoplasm. Colocalization was defined as an area of overlap between two foci of different fluorophores. The maximum number of foci that could be counted in these cells was 150. For purposes of quantification of γ-H2AX foci in the γ-H2AX-bright cells, the γ-H2AX-bright cells were assigned 151 foci, which was one focus more than the maximum countable number. A typical immunofluorescence experiment consisted of assessment of 30–50 cells per condition. The data presented are from two to three independent experiments performed on two or three clones of each type. Immunofluorescence images for figures were created using Image J and Metamorph software (Molecular Devices).A focus was defined as a defined area of the nucleus greater than the minimum area of optical resolution  at a concentration of approximately 5×106/ml. Cells were incubated overnight at 4°C in Alexa Fluor 647–conjugated anti-H2A.X (BioLegend). After incubation, the cells were washed twice with 0.1% saponin and 5% fetal bovine serum in PBS and were resuspended in 5% fetal bovine serum in PBS for analysis. Cells were analyzed on a FACScaliber as described above. Data were collected from a minimum of two experiments on five BLM+ clones and six SM-BLM clones. Median fluorescence intensity data was log normalized, and the differences in median intensity were calculated. Log-normalized median intensity differences were tested by Student's t-test.Cells were untreated or treated with 0.5 mM HU for 24 h. Immediately after treatment, cells were harvested, washed twice with PBS, resuspended in fixation solution  at a density of approximately 1×105 cells were formed into individual 1% agarose plugs . The plugs were then incubated in 100 mM EDTA (pH 8.0), 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, and 1 mg/ml proteinase K at 50°C for 24 h. The plugs were washed four times in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA for 30 min at room temperature with gentle agitation. Plugs were loaded onto a 0.8% agarose gel , and PFGE was preformed on a CHEF DR III  for 72 h. The gel was stained with SYBR Gold, visualized under UV light, and analyzed using Quantity One and ImageJ software after contrast adjustment. Each lane on the gel was divided into seven areas, and the intensity in each area was analyzed and weighed according to fragment size. The amount of breakage in each lane was further normalized against total DNA content. Values are given relative to the level of DNA breakage in the untreated control. At least two independent experiments were performed on two clones of each type.Cells were untreated or treated with 0.5 mM HU for 24 h and subsequently released into fresh medium for an additional 0, 12, or 24 h, or they were treated with different concentrations of CPT for 3 h. For each damage condition, 4×10For SCE analyses, cells were cultured with 10 µM BrdU (Sigma-Aldrich). After 60 h, the cells were incubated with 0.02 µg/ml colcemid (Invitrogen) for up to 2 h, harvested and processed as described earlier To quantify the cytogenetic effects of replication-associated DSBs, cells were treated with HU, and micronuclei formation was assessed using the cytokinesis-block micronucleus assay 5 cells were seeded onto six-well dishes. Cells were treated or not treated with 0.5 mM HU for 24 h and then treated or not treated with 50 µM of etoposide for an additional 24 h. At the end of the second treatment, the cell-growth medium was retained, and the floating cells were combined with adherent cells harvested by trypsinization. We added the ViaCount reagent (Guava Technologies) according to the manufacturer's instructions. Live and dead cells were counted with the Guava Cell Analyzer. A minimum of three independent experiments with three replicates for each condition were performed on three clones of each type.To measure cell viability under different DNA damage conditions, 2×10Human RAD51 protein was purified as described previously BLM N-terminal fragment (1–431) was modified by SUMO-2 in vitro as described Because observations within each clone may be correlated, we used mixed effects linear models to test the data for statistical significance. In the mixed effects models, each clone was treated as a random effect, and the experimental variables were treated as fixed effects. Because the foci and micronuclei data were not normally distributed, we first applied a square root transformation to stabilize the variance and normalize the data . For testing changes in the number of foci per cell and the number of colocalized foci per cell, cell type (BLM or SM-BLM), treatment (with and without HU), and interaction terms for cell type by treatment were treated as fixed effects. Similarly, for testing changes in the number of micronuclei per cell and the number of SCEs per 46 chromosomes, cell type, treatment with HU, and their interaction were treated as fixed effects. Finally, for testing changes in percentage of cell death, cell type, treatment with HU, treatment with etoposide, and their interaction terms were included as fixed effects. If the random effects  were found to be nonsignificant based on likelihood ratio test, the mixed effect models reduced to traditional analysis of variance.Figure S1BLM+ and SM-BLM cells have similar cell-cycle profiles. Representative cell-cycle profiles of cells expressing BLM or SUMO-mutant BLM after HU treatment as determined by BrdU incorporation and flow cytometry analysis. Cells were untreated (NT) or treated with 0.5 mM HU for 24 h. Cells were then analyzed at 0 h, 1 h, 3 h, and 6 h after release from the HU block. Greater than 80% of the cells were in S phase after release from the HU block.(0.42 MB PDF)Click here for additional data file.Figure S2BS cells accumulate excess DSBs at damaged replication forks. Quantification of DSBs in untreated BS cells (GM08505) (NT) or cells treated with 0.5 mM HU for 24 h, followed by release into normal medium for 0, 12, and 24 h. Bars represent the numbers of DSBs relative to untreated BS cells in three independent experiments. Error bars represent standard deviation of the data.(0.01 MB PDF)Click here for additional data file.Figure S3SM-BLM cells are hypersensitive to camptothecin (CPT) compared to BLM+ cells, as determined by colony survival assays. A total of 1,000–25,000 cells were seeded onto six-well plates overnight, untreated or treated with different concentrations of CPT for 3 h. After treatment, cells were allowed to form colonies in normal medium. Percentage colony survival was calculated as [number of colonies]treated/[number of colonies]untreated×100. Data represent a single experiment performed on five BLM clones and four SM-BLM clones. GM08505 is the parental BS SV40-transformed human fibroblast cell line and GM00637 is a normal SV40-transformed human fibroblast control; the experiment was performed two times for these cell lines.(1.88 MB PDF)Click here for additional data file.Figure S4BS cell phenotypes differ from SM-BLM cell phenotypes. (A) HU induces increased levels of SCEs in BS cells. BS cells (GM08505) were incubated with 10 µM BrdU for 30 h, treated or not with 0.5 mM HU for 24 h, and then returned to BrdU-containing medium for an additional 20 h. Metaphases were collected in colcemid. Two photomicrographs are shown of metaphases in which exchanges between sister chromatids were visualized. Average numbers of SCEs/46 chromosomes for untreated and HU-treated BS cells is shown beneath the photomicrographs (number of metaphases counted). (B) HU-treated BS cells (GM08505) exhibit excess colocalization of RAD51 and γ-H2AX, demonstrating that RAD51 is effectively recruited to damaged replication forks in the absence of BLM. Bars indicate 10 µm.(5.80 MB PDF)Click here for additional data file.Figure S5Graphical representation of mean numbers of colocalized RAD51 and BLM foci in untreated (NT) and HU-treated (HU) BLM+ and SM-BLM cells.(0.01 MB PDF)Click here for additional data file.Figure S6Impaired localization of RAD51 to damaged forks in SUMO-mutant BLM cells after a short treatment with HU. BLM+ and SM-BLM cells were untreated (NT) or synchronized with 0.5 mM mimosine for 24 h, which stalls cells in late G1 phase, released into normal medium for 5 h to allow the cells to enter S phase, then treated (MIM+HU) or not (MIM rel) with 10 mM HU for 1 h and evaluated for RAD51 localization with BLM and γ-H2AX. BrdU flow cytometry, as performed in (0.01 MB PDF)Click here for additional data file.Figure S7BLM and SM-BLM localize to stalled replication forks with similar efficiency. (A) Immunofluorescence images of representative S-phase BLM+ and SM-BLM cells treated with 0.5 mM HU for 24 h. Cells were stained with antibodies to PCNA. Images show GFP-BLM fluorescence, PCNA staining, and merged BLM-PCNA immunofluorescence. (B) Graphical representation of the average numbers of BLM foci, PCNA foci, and colocalized BLM-PCNA foci in HU-treated BLM+ and SM-BLM cells. The numbers of BLM and PCNA foci were counted in cells that stained positively for PCNA, as these represented cells in S phase. Data presented are the average of two experiments in each of two BLM+ and two SM-BLM clones. Bars represent the mean numbers of foci or colocalized foci per nucleus, and the error bars represent the standard deviations of the combined data. The levels of BLM foci and PCNA foci were higher in SM-BLM than in BLM+ cells; however, the percentages of colocalized BLM-PCNA foci were similar in BLM+ and SM-BLM cells, indicating that SUMO-mutant BLM localizes normally to sites of replication damage.(0.99 MB PDF)Click here for additional data file.


Cerebral state monitor is a monitor which shows depth of anesthesia in a number between 0–100 as cerebral state index, in which 40–60 is appropriate for general anesthesia. The effect of electromyogram on cerebral state index has not been shown yet.A 24-year-old Iranian-balooch man admitted in the intensive care unit because of head injury in a car accident. In spite of sustained low level of consciousness, his cerebral state index had significant fluctuations coordinated with electromyogram resulted from facial muscle contractions. After neuromuscular blocking agent prescription, cerebral state index was decreased from about 90 to 40, directly followed the changes in electromyogram. The Cerebral State Monitor  is a portable, wireless monitor that uses the time and frequency domain analysis, which inputs into a fuzzy logic inference system to show a 0 to 100 scale, the Cerebral State Index (CSI), with 40 to 60 indicating an adequate depth of hypnosis . MoreoveA 24-year-old Iranian-balooch man was admitted in the ICU because of head injury. He had not any history of medical or surgical diseases before the accident. His CT scan showed generalized brain edema, the Glasgow coma scale score (GCS) on arrival was 4. In the ICU the patient was sedated by remifentanil and midazolam to decrease the brain edema via hyperventilation and decreasing blood pressure. Twenty four hours later, the CSI was appeared with fluctuations that correlated to muscular twitching in the facial muscles. The CSI had also fluctuations parallel to EMG activities which appeared on the CSM screen with a short time delay, less than one minute figure  . When EThe aim of the Cerebral State Index (CSI) is to monitor the level of consciousness or hypnosis during general anesthesia or in the ICU. The CSI is a unitless scale from 0 to 100, where 0 indicates a flat electroencephalographic signal and 100 indicates the awakening state. The 40–60 range is adequate range for anesthesia. The CSI requires three electrodes positioned at the middle forehead, left forehead, and left mastoid. Alternatively, the right forehead and right mastoid can be used.The CSI is calculated based on four sub parameters of the electroencephalogram:Alfa ratio, Beta ratio, Alfa ratio – Beta ratio, and burst suppression, calculating an index from 0 to 100. The novelty of the CSI is that a fuzzy inference system was used in Inference System (ANFIS). During burst suppression, the Alfa and Beta ratios are no longer monotonously decreasing as a function of anesthetic depth, and therefore, they cannot be used in the calculation of the final index . CalculaIn conclusion, CSI can be used for detecting depth of anesthesia or sedation, but overlapping EEG with EMG is an important and sometimes very hazardous pitfall. Wrong assessment of depth of anesthesia can lead to administrate so many anesthetic or hypnotic drugs with many life threatening adverse effects in operating theatre, or misinterpretation of the brain condition of the patient causes premature weaning of the sedation and ventilatory support of the patient.CSI: cerebral state index; CSM: cerebral state monitor; BIS: bispectral indexl; EMG: electromyogram; BS: burst suppressionl; EEG: electroencephalogram.The author declares that they have no competing interests.The single author was involved in the management of the case and finalizing the article. The single author was involved in the process of editing, correcting, and finalizing the manuscript. The author has read and approved the final manuscript.Written informed consent was obtained from the patient's family for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The utility of current chemotherapeutic regimens in the treatment of AIDS-related Kaposi's sarcoma (AIDS-KS) is often compromised by both limited efficacy and substantial toxicity. Pegylated  liposomal doxorubicin hydrochloride (SL-DOX) has been demonstrated specifically to deliver high concentrations of doxorubicin to Kaposi's sarcoma (KS) lesions. This phase II study was performed to evaluate the efficacy and safety of SL-DOX in the treatment of moderate to severe AIDS-KS. Patients were treated biweekly with 10, 20, or 40 mg m-2 SL-DOX. Tumour response was assessed according to AIDS Clinical Trials Groups (ACTG) criteria before each cycle. Best response was determined for 238 patients and was achieved after a mean of 2.3 cycles (range 1-20). Fifteen patients (6.3%) had a complete response to SL-DOX, 177 (74.4%) had a partial response, 44 (18.5%) had stable disease and two (0.8%) had disease progression. SL-DOX was well tolerated: ten patients discontinued therapy because of adverse events, in four cases because of neutropenia. Grade 3 or 4 neutropenia occurred after 281 of 2023 cycles (13.9%) but involved 137 of 240 patients (57.1%) for whom data were available. SL-DOX has substantial activity in AIDS-KS. Best response is typically seen after fewer than three cycles of chemotherapy and in some cases may be prolonged. The most important adverse event is neutropenia, which occurs after a minority of cycles but which may occur in over half of all patients.


While access and utilization form core components in assessing the effectiveness of a health service, the concept of coverage is often neglected. In this study we propose to develop a GIS-based methodological framework for the measurement of district-based geographic coverage to examine the service effectiveness of methadone treatment programme (MTP) in Hong Kong on a regular basis.To overcome the incompatibility of spatial units, population data and data of heroin addiction of the year 2001 are interpolated by population-weighted and area-weighted algorithms. Standard overlay and proximity analytical functions are used to delineate altogether 20 accessible zones around each methadone clinic at a fixed 1.5 km Euclidean distance. Geographic coverage here is defined as the percentage of heroin addicts covered by a methadone clinic within the accessible zone by district.A total of 6413 out of 11000 reported heroin addicts are found geographically covered. The average geographic coverage in Hong Kong is 44.6%, with the figure varying from 0% to 96% by district. One district having no clinic results in 0% coverage whereas another without a clinic yields 15.3% coverage from the clinic in adjacent district. Maps illustrating district-based geographic coverage are generated.As continuous data collection is required for a monitoring system, the simplified approach facilitates the handling of large volume data and relevant data analysis. It is concluded that the number of methadone clinics is as important as their locations. Geographic coverage could become an important consideration for monitoring harm reduction. Methadone maintenance is one of the well-known harm reduction strategies for public health intervention in heroin addiction. The significance of methadone treatment in preventing needle sharing, which in turn reduces the risk of HIV and HCV transmission among injectors, has been well cited. -4 MethadTo achieve maximum effectiveness, methadone clinic should be situated at where it satisfies the needs of local community of heroin users. The identification of the service zone of a health service and an examination of whether it is reachable are crucial. In this connection, the concept of coverage is useful. This is particularly meaningful for evaluating the provision of methadone maintenance programme, a service that delivers public health good. While large proportion of studies to date have focused on access and utilization of health care systems -10, few In this study, Hong Kong is taken as an example to pilot the measurement of the district-based geographic coverage of methadone clinics in terms of their availability within defined accessible catchment areas. The study aims at developing a simplified methodological framework to measure the geographic coverage of methadone clinics regularly, which enables a monitoring system to be developed for evaluating harm reduction strategies.Hong Kong is a metropolitan city located in Southeast Asia, bordering the South China Sea and mainland China . The totThe MTP has been operated by the Department of Health  for 30 years. It adopts an out-patient and low threshold approach. Services are provided promptly to all heroin addicts who come forward for methadone treatment. For those with life-threatening medical condition, referral for treatment is made before being started on methadone. Besides prescribing methadone, other related services including patient assessment, counselling services and referral services are provided in methadone clinics . CurrentEssential data for this study included population data and data of heroin addiction. All data obtained were of the year 2001, when consistent data types from different services could be assured. As a study to demonstrate the methodological framework of coverage measurement, the use of the 2001 data, instead of more updated but inconsistent datasets, would be more important to enable reliable calculations to be made. District-based and TPU-based population data were collected from the Census and Statistics Department, while district-based data of heroin addiction was acquired from the Central Registry of Drug Abuse (CRDA), Narcotics Division of Security Bureau. CRDA has been established for 35 years to capture drug abuse data, which are provided by the reporting agencies on a voluntary basis with their full cooperation. The statistics is used to monitor the trend of drug abuse and to facilitate the planning of drug rehabilitation programmes in Hong Kong.Digital base maps including the boundary map of districts, boundary map of TPUs, map of highlands and water bodies were obtained from the Survey and Mapping Office (SMO), Lands Department of Hong Kong Government. Locations of methadone clinics were stored as X, Y projected coordinates in the GeoCommunity Database table (Version 2.1.2), which was also acquired from the SMO. These data, after synchronization, were input into a GIS software, ArcGIS 9.1 , for datThe landscape in Hong Kong is mountainous. About one-fifth of land area is over 200 metres in altitude, which is defined as uninhabitable highland in this study. Furthermore, the result will be highly unreliable if heroin addicts are assumed to distribute over the entire district, considering that only part of the district is populated. District-based heroin addicts' data were apportioned into TPUs by areal interpolation before measuring district-based geographic coverage. From the interactions with current and former heroin users, three assumptions for this study were made. First, walking is a preferred means of going to methadone clinics. Second, heroin addiction is assumed to be rather localized. Heroin addicts seldom go to the methadone clinic that is not located in their residence district. Third, heroin addicts are willing to walk at most for 30 minutes to their local methadone clinics.In preparing the Hong Kong base maps for spatial analysis, all water bodies, including reservoir and rivers, and land elevated over 200 metres were eliminated.  is proportional to its population, sharing the same ratio of heroin addicts by district over population by district. Figure  Thus theNumber of heroin addicts by district / population by district * population by a specific TPUEuclidean distance was defined as accessible zone for each clinic, assuming that addicts will cover this distance in 30 minutes.  statements were used in the GIS to calculate both the area of entire intersected TPUs and the area of intersected portion of TPU. Percentage of area covered by the accessible zones in each intersected TPU was calculated and TPU-based heroin addicts covered were estimated by using the percentage. The following equation was used:Area of a TPU within the accessible zone / Area of the entire corresponding TPU* Interpolated number of heroin addicts in that TPUNumber of addicts from all TPUs in a district covered within accessible zones was aggregated into their corresponding district. However, the number of methadone clinics in each district varied considerably. The number of district-based addicts covered was adjusted and the mean number of heroin addicts covered by one methadone clinic by district was calculated. District-based geographic coverage was expressed as the percentage of heroin addicts covered within those accessible zones in each district among all heroin addicts in that district, using the following formula:Number of heroin addicts covered within a accessible zone in a district / Total number of heroin addicts in that corresponding district* 100%In the year 2001, there were about 11000 reported heroin addicts, giving a prevalence of 166 heroin addicts per 100,000 population in Hong Kong. The prevalence in eight out of 18 districts was higher than the average, with Yau Tsim Mong yielding the highest rate of 403. The distribution of methadone clinic was uneven. While one district was having three methadone clinics, two districts including Kwai Tsing and Sai Kung did not have any. Most of the districts (13 out of 18) had one methadone clinic, with two districts having two. After the elimination of uninhabitable areas, all TPUs (N = 197) remained populated and were therefore valid for conducting areal interpolation. The population-weighted areal interpolation produced interpolated values of heroin addicts by TPUs ranging from 1 to 369, with one-forth of TPUs (50/197) having less than 10 heroin addicts. Though all TPUs could be subjected to population-weighted areal interpolation, only 125 of them gave an intersection with the defined accessible zones for a clinic. In fact, an average of 66% of TPUs in a district was covered by the accessible zones. Accessible zones in four districts even covered all TPUs in that particular district.Using the definition developed in this study, a total of 6413 of 11000 heroin addicts were geographically covered by all methadone clinics in 2001. Since there were more than one methadone clinic in some districts, adjusted number of heroin addicts covered in each district was calculated. Only one district, Sai Kung, showed no coverage by any methadone clinic. Geographic coverage varied from the lowest 15.3% in Kwai Tsing to the highest figure 96.2% in Yau Tsim Mong. In the remaining districts, the average geographic coverage was 44.6% in 2001, though eight out of 18 districts demonstrated over 50% geographic coverage.  declare that they have no competing interests.SSL was involved in the conceptualization and research design of the study. PTTP conducted the cartographic design and GIS analyses. This manuscript was drafted by PTTP and edited by SSL. Both authors read and approved the final manuscript.


Sir,The recent publications of On a further point,


A new hybrid material consisting of a polyhedral oligomeric silsesquioxane (POSS) and carbon nanotube (CNT) was synthesized by a simple and versatile approach entailing click coupling between azide moiety-functionalized POSS and alkyne-functionalized multi-walled CNTs. This approach provides a simple and convenient route to efficiently functionalize a wide variety of nanoscale nanostructure materials on the surface of CNTs. A hybrid nanomaterial can be broadly depicted as a multi-component system where two or more nanomaterials are unified to form a new nanomaterial fabricated with the aim of realizing attractive multi-functional properties. Hybrid nanomaterials of carbon nanotubes (CNTs) with metals, metal oxides, and biological compounds have been developed for various applications such as sensors, actuators, solar cells, biosensors, and light emitting devices ,2. CNTs Polyhedral oligomeric silsesquioxane (POSS), a type of inorganic nanostructured molecule -9, contaThis article describes the synthesis of a CNT-POSS nanohybrid material using a click chemistry reaction. It is anticipated that this approach can be utilized to prepare nanohybrids with high interfacial bonding.p-nitrophenol, terabutylammonium bromide, 3-methyl butyl nitrite, copper iodide, and 1,8-diazabicyclo[Multi-walled carbon nanotubes (MWNTs) used in this study were purchased from Iljin Nano Tech, Seoul, Korea. Their diameter and length ranges were approximately 10-20 nm and 20 μm, respectively. EP0402-epoxycyclohexyllsobutyl POSS , propargyl bromide, zabicyclo,4undecen1H NMR and 13C NMR spectra were measured on a 400-MHz instrument by Bruker on CDCl3 solutions at room temperature. Raman spectroscopy  was used to confirm the functionalization of MWNTs. X-ray photoelectron spectroscopy  was used to analyze the surface composition of the nanotubes. Observation of the surface morphology and energy dispersive X-ray spectrum (EDX) measurement of the MWNT-POSS nanohybrid was carried out by transmission electron microscopy . Thermogravimetric analysis (TGA) was carried out in a TA Q 50 system TGA.Fourier transform-infrared (FT-IR) spectroscopic measurements were performed using a Jasco FT-IR 300E device. Elemental analysis was determined by Perkin-Elmer analyzer model 2400 CHN analyzer. p-aminophenyl propargyl ether was first synthesized according to a procedure reported in the literature [p-aminophenyl propargyl ether were placed in a two-necked flask fitted with a reflux condenser and a magnetic stirrer bar under a N2 atmosphere. Then, 3.0 g 3-methyl butyl nitrite was slowly injected via a syringe, and the reaction mixture was stirred at 60°C for 5 h. The resulting product was washed three times with 100 ml of dimethylformamide (DMF), and dried under vacuum at 60°C for 80 h, and the product yield was 80%.For the click reaction, terature  to introThe azidation of the POSS molecule was carried out with sodium azide in the presence of ammonium chloride, as shown in Figure 3 in 15 ml of DMF. The flask was equipped with a magnetic stirrer bar with a reflux condenser. 162 mg (0.85 mmol) of copper iodide and 6.4 g (42.5 mmol) of 1,8-diazabicyclo[Coupling of an azide moiety-containing POSS and alkyne-functionalized MWNTs was carried out via Cu(I)-catalyzed click chemistry. Typically, 20 mg of alkyne-functionalized MWNTs was dispersed in 15 ml of DMF. The MWNTs solution was added to a two-necked flask containing a 400 mg (0.43 mmol) solution of POSS-N3) and alkyne-functionalized MWNTs  with azide-functionality. A new peak at 2107 cm-1 corresponding to the azide group [-1 for OH stretching were observed. The results of 1H NMR and 13C NMR measurements reveal clearly the POSS-N3 structure  and 1585 cm-1 (G band) [sp3-hybridized carbons of the nanotubes, whereas the G band corresponds to a splitting of the E2g stretching mode of graphite, which reflects the structural intensity of the sp2-hybridized carbon atoms. The increase in the band intensity ratio (ID/IG) of the functionalized MWNTs reflects the relative degree of functionalization or defects in the nanotubes, indicating covalent functionalization MWNT-POSS nanohybrids. TEM images of the MWNT-POSS nanohybrid  . The D bd Figure  show thad Figure .3 molecule reacted with alkyne-functionalized MWNTs. The atomic percent and weight percent of Si for the MWNT-POSS nanohybrid were calculated by EDX measurment as 3.98 and 8.57%, respectively -catalyzed azide-alkyne cycloaddition between azide moiety-containing POSS and alkyne-functionalized MWNTs. Click coupling can provide a new strategy for the synthesis of CNT-based nanohybrids.CNT: carbon nanotube; DMF: dimethylformamide; EDX: energy dispersive X-ray spectrum; FT-IR: Fourier transform infrared; MWNTs: multi-walled carbon nanotubes; POSS: polyhedral oligomeric silsesquioxane; TEM: transmission electron microscopy; TGA: thermogravimetric analysis; THF: tetrahydrofuran; XPS: X-ray photoelectron spectroscopy.The authors declare that they have no competing interests.SKY conducted all the experiments and drafted the manuscript. SSM helped in technical support for experiments and characterization. HJY participated in measurements and data analysis. JWC designed the experiments and supervised the all of the study. All the authors discussed the results and approved the final manuscript.


To identify determinants associated with disease progression and death following human immunodeficiency virus (HIV) diagnosis.Disease progression data from the diagnosis of HIV infection or acquiring immunodeficiency syndrome (AIDS) to February 29, 2012 were retrospectively collected from the national surveillance system databases and the national treatment database in Wuhan, China. Kaplan-Meier method, Logistic regression and Cox proportional hazards model were applied to identify the related factors of progression to AIDS or death following HIV diagnosis.HR) for progression to AIDS following HIV diagnosis . HIV infectors at least 60 years presented 1.15-fold  increased risk to develop AIDS when compared with those aged 17–29 years. Similarly, AIDS patients with diagnosis ages between 50 and 59 years were at a 1.60-fold higher risk of death  compared to those aged 19–29 years. AIDS patients with more CD4+ T-cells within 6 months at diagnosis (cell/µL) presented lower risk of death . The highly active antiretroviral therapy (HAART) delayed progression to AIDS from HIV diagnosis  and reduced the risk of death after AIDS diagnosis .By the end of February 2012, 181 of 691 HIV infectors developed to AIDS, and 129 of 470 AIDS patients died among whom 289 cases received concurrent HIV/AIDS diagnosis. Compared with men infected through homosexual behavior, injection drug users possessed sharply decreased hazard ratio (+ T-cell counts and HAART. Effective interventions should target those at higher risk for morbidity or mortality, ensuring early diagnosis and timely treatment to slow down the disease progression.Progression to AIDS and death following HIV diagnosis differed in age at diagnosis, transmission categories, CD4 Since China’s first acquired immunodeficiency syndrome (AIDS) case was identified in a dying tourist in 1985 In order to provide HIV treatment and care primarily for rural and low-income urban patients who met the national treatment guidelines, the National Free Antiretroviral Treatment Program (NFATP) was initiated in October 2002 and subsequently expanded nationwide in 2003 + T-cell count Despite this dramatic achievement, there remain questions about whether survival differs in specific subpopulations and what factors may drive such variation. Previous studies have suggested that age, gender, transmission category, educational level, marital status, and CD4+ T-cell count <200 cells/µL (increasing to <350/µL in 2008), or total lymphocyte count <1200 cells/µL. HIV infectors and AIDS patients were followed up with a face-to-face interview every six months and three months, respectively. After each visit, local health care workers completed a standardized case report form (CRF) and faxed the form to the Chinese CDC via Datafax  Subjects recruited in this study were identified through the two national databases, namely national surveillance system and national treatment database. In China, all HIV/AIDS cases diagnosed are compulsorily reported to CDC through the national surveillance system, which was upgraded to China Information System for Disease Control and Prevention (CISDCP) in 2003 The written consents were obtained from the Wuhan Center for Disease Control and Prevention which declared their willing to participate in the study and the data relating to disease progression of HIV/AIDS cases can be extracted from the national surveillance system databases and the national treatment database. All information was de-identified and only aggregated data were used for data analysis. The study was approved by the institutional review board of Tongji Medical College of Huazhong University of Science and Technology.We used data to determine disease progression to AIDS following HIV diagnosis among adolescents and adults (≥13 years old at diagnosis). We included subjects who (1) were Wuhan residents and had been reported by Wuhan CDC, (2) were confirmed to be HIV-infected by a positive Western blot result, and (3) were aged older than 13 years at diagnosis. Given difference in disease progression between adolescents and adults, we excluded infectors who were infected perinatally or younger than 13 years of age at diagnosis + T-cell count <200 cells/µL before February 29, 2012 were included. Patients were followed up from the date of AIDS diagnosis until February 29, 2012. Individuals free of death at censoring date were assumed to be alive. Patients who died not due to AIDS or AIDS-defining illnesses during the follow-up period were censored at date of death. Of 472 AIDS cases met the criteria above, 2 (0.42%) were excluded from the analyses because of too much missing information.We also determined survival among adolescents and adults (≥13 years old) diagnosed with AIDS. Subjects who met the inclusion criteria in the part of HIV diagnosis to AIDS, and received a diagnosis of AIDS either by an AIDS-defining illness or by having a CD4The primary end point of the AIDS-related mortality was death attributable to AIDS or AIDS-defining illnesses. By using the overall period 1999–2002 as a whole owing to the small numbers of annual AIDS-related death during these years, then yearly from 2003 to 2011, the AIDS-related mortality was defined as the total number of subjects who died of AIDS or AIDS-defining illnesses within the specified time period divided by the sum of person-years for individuals who received a diagnosis of AIDS during the same period. The year that a person was lost to follow-up or died of other causes  was counted as one-half of a person-year. A similar method was conducted to calculate each individual’s person-years receiving HAART during each time period. The Log-linear model was used to determine whether a decreasing trend for mortality existed.+ T-cell count at AIDS diagnosis (first record within 6 months at diagnosis), and concurrent diagnosis of HIV diagnosis and AIDS or not (only for AIDS cases). The cross-table was applied to assess the linear trend in morbidity and mortality among strata of selected variables.Kaplan-Meier method was used in STATA software  to calculate the proportions of HIV infectors who progressed to AIDS and relative survival proportions for AIDS patients. Stratified analyses were conducted by sex, age at AIDS diagnosis or HIV diagnosis, transmission category, marital status, educational level, receiving HAART or not, CD4α = 0.05.We used non-conditional Logistic regression analysis and Cox proportional hazard model to address potential risk factors of AIDS morbidity and mortality As reported to the Wuhan CDC, 691 persons received HIV diagnosis from 1994 to February 2012, of whom 181 HIV infectors developed to AIDS. 11 (1.59%) HIV infectors were lost to follow up. 456 (65.99%) HIV infectors did not progress to AIDS and were still alive, while 43 (6.22%) cases died before they progressed to AIDS. The majority of HIV infectors were male (85.14%) and attained secondary education (60.20%). 679 (98.26%) HIV infectors were Han Chinese, and the other 12 were minorities. The median age at HIV diagnosis was 34 years old, ranging from 17 to 85 years old [interquartile range (IQR): 26–46 years old]. Men who had sex with men (MSMs) accounted for 48.63%, followed by heterosexual contact (HC) with someone at high risk or with a diagnosis of HIV infection or AIDS, injection drug users (IDUs), infection through Blood (blood transfusion or blood products), and other unknown reasons . The sinThe mean follow-up time of HIV infectors was 18.58±20.16 months. 17.90%, 28.15% and 57.27% of HIV infectors progressed to AIDS after one year, two years, and five years, respectively. The rate of progression to AIDS within one or three years following HIV diagnosis was similar between males and females, and it was lower for individuals receiving HAART than those did not. The proportion of HIV infectors with progression to AIDS presented no difference in age at diagnosis, transmission categories, marital statuses, educational level within one year following diagnosis. Otherwise, different proportion of HIV infectors with progression to AIDS arose among the groups mentioned above except educational level at three years .P<0.05 for all). After mutual adjustment by these factors, sex, age at diagnosis, and marital status were no longer statistically significant associated with AIDS events . Otherwise, HIV infectors transmitted by HCs and Blood were more likely to develop AIDS compared to MSMs . The more educated HIV infectors and those under HAART were less likely to develop AIDS .Results of univariate and multivariate non-conditional Logistic regression analyses are presented in P for trend = 0.048), whereas education turned out to be a protective factor (P for trend <0.001). The results of the multivariate analysis after adjustment for sex and factors statistically significant in univariate analysis showed that IDUs were less likely to develop AIDS following HIV diagnosis  compared with MSMs. The individuals aged ≥60 years had a 1.15-fold higher risk of progressing to AIDS  compared with those aged 17–29 years. The HAART reduced substantially the risk of progression to AIDS .+ T-cell counts (<50 cells/µL) accounted for 23.4%, and 68.94% of patients received HAART.470 persons received AIDS diagnosis from 1999 to February 2012, of whom 129 died, including 27 censored patients who died not due to AIDS or AIDS defining diseases. 5 (1.06%) AIDS patients were lost to follow up, and 336 (71.49%) AIDS patients survived. 289 of 470 (61.46%) AIDS patients received concurrent HIV/AIDS diagnosis. The majority of AIDS patients were male (75.32%) and attained secondary education (66.38%). 454 (96. 06%) AIDS patients were Han Chinese and the other 16 were minorities. The median age at AIDS diagnosis was 39 years old, ranging from 19 to 79 years old (IQR: 31–49 years old). The married patients accounted for 42.98%, and over half (52.12%) of the patients with AIDS were infected through HC . AIDS PaP<0.05). Conversely, the percentage of AIDS patients receiving HAART increased from 2.56% of person-years in 2003 to 53.33% in December 2011, presenting an inverse relationship with AIDS-related mortality.The AIDS-related mortality over time among AIDS patients are shown in + T-cell counts within 6 months at diagnosis (cell/µL), concurrent HIV/AIDS diagnosis and receiving HAART . After mutual adjustment by these factors, sex, age at diagnosis, transmission categories, marital status and concurrent HIV/AIDS diagnosis were no longer statistically significant associated with death events . Otherwise, the more educated AIDS patients and those under HAART were less likely to die of AIDS or AIDS-defining illnesses . Patients with more CD4+ T-cell count within 6 months at diagnosis was associated with less likelihood to die of AIDS or AIDS-defining illnesses .Results of univariate and multivariate non-conditional Logistic regression analyses are presented in + T-cell count within 6 months at diagnosis, concurrent HIV/AIDS diagnosis, and HAART, had significant influence on AIDS survival. The results of the univariate analysis indicated significant differences among age at diagnosis, transmission categories, marital statuses, educational level, CD4+ T-cell count within 6 months at diagnosis, concurrent HIV/AIDS diagnosis, and HAART. Age might be a risk factor for death (P for trend = 0. 01), whereas education turned out to be a protective factor (P for trend <0.001). The risk of death was inversely related to the number of CD4+ T-cells within 6 months at diagnosis (P for trend <0.001). The results of the multivariate analysis after adjustment for factors statistically significant in univariate analysis (plus sex) showed that AIDS patients with diagnosis ages between 50 and 59 years presented a 1.60-fold higher risk of death  compared with those aged 19–29 years. With regard to CD4+ T-cell count within 6 months at diagnosis, AIDS patients with more number of CD4+ T-cells within 6 months at diagnosis had lower risk of death . The HAART reduced substantially the risk of death .+ T-cells within 6 months at diagnosis (cell/µL). HAART reduced the risk of progression to AIDS after HIV diagnosis and death after AIDS diagnosis.Quantification of the survival after AIDS and identification of characteristics present at HIV or AIDS diagnosis offered valuable information for the better understanding about the progression of HIV infection. Our study addressed such issues and found that transmission categories and age at diagnosis were related to progression to AIDS after HIV diagnosis. Besides, Survival after AIDS diagnosis differed among age at AIDS diagnosis and number of CD4P<0.05), compared with that in 2003, which was mostly attributable to the NFATP From the surveillance data, 74.86% of the Wuhan native AIDS patients survived 5 years after AIDS diagnosis. The one-year-survival, two-year-survival and five-year-survival were higher than that in a study conducted in Italy  + T-cell count at AIDS diagnosis). Consistently with previous evidence, disease severity was proved as an independent risk factor for poor survival The findings of the multivariate Cox proportional hazards regression analysis revealed the potential disparities in disease progression among persons with an HIV or AIDS diagnosis. We found that excess mortality risk exist in AIDS patients with more severe disease  version + T-cell count<200 cells/µL or by an AIDS-defining illness. 32.87% AIDS patients had very low CD4+ T-cell counts at AIDS diagnosis (<50 cells/µL). HIV diagnosis at advanced disease may indicate rapid disease progression or late diagnosis in the disease process (testing may be motivated by symptoms). Because of social or economic factors, HIV infectors might be concurrently diagnosed with AIDS at low CD4+ T-cell counts for lack of adequate care to monitor the infection and institute treatment In the first edition (2005) of the China Free ART Manual, the CD4HR = 1.49; 95% CI, 1.34–1.66 per 10-year increase) was found in the UK population in 2008 + T-cell count or viral load, older patients had a greater risk for progressing to AIDS than younger ones. However, adherence to treatment appeared to be higher among older compared with younger HIV infectors Older patients presented increased risk for disease progression in our analyses, which might be caused by diminished immune function and recovery and higher comorbidity Decreased educational level was associated elsewhere with increased mortality Previous studies investigated delayed diagnosis of HIV by using varying time frames (ranging from AIDS diagnosis within one to twelve months of HIV diagnosis) + T-cells counts) and the virus load at AIDS diagnosis for many cases (data not shown), which might affect our results Since our study was based on retrospective data, there were inevitable some limitations. Firstly, we did not have information on severity of disease at HIV diagnosis (CD4+ T-cell counts. HAART had significantly delayed the progression to AIDS following HIV diagnosis and improved the survival of AIDS patients. Thus effective interventions should target those at higher risk for morbidity or mortality, ensuring early diagnosis and timely treatment to slow down the disease progression. In future, a prospective study with larger size of sample should be carried out to obtain more information about the AIDS progression.In summary, progression to AIDS and death following HIV diagnosis differed in age at diagnosis, transmission categories and CD4


Ranavirus, and in the past few decades, FV3 infections have resulted in considerable morbidity and mortality in a range of wild and cultivated amphibian species in the Americas, Europe, and Asia. The reasons for the pathogenicity of FV3 are not well understood.Frog virus 3 (FV3) is the type species of the genus r, and reported that our wt-FV3 and aza-Cr strains showed similar levels of virulence, while SSME was the least virulent in an in vivo study with Lithiobates pipiens tadpoles. Using 454 GS-FLX sequencing technology, we sequenced SSME and compared it to the published wt-FV3 genome. SSME had multiple amino acid deletions in ORFs 49/50L, 65L, 66L, and 87L, which may explain its reduced virulence. We also investigated repeat regions and found that repeat copy number differed between isolates, with only one group of 3 isolates and 1 pair of isolates being identical at all 3 locations.We investigated three FV3 isolates designated SSME, wt-FV3, and aza-CIn this study we have shown that genetic variability is present between closely related FV3 isolates, both in terms of deletions/insertions, and even more so at select repeat locations. These genomic areas with deletions/insertions may represent regions that affect virulence, and therefore require investigation. Furthermore, we have identified repeat regions that may prove useful in future phylogeographical tracking and identification of ranaviral strains across different environmental regions. Batrachochytrium dendrobatidis[Iridoviridae have also been associated with amphibian decline. The family Iridoviridae is comprised of large, cytoplasmic, double stranded DNA viruses with icosahedral capsids [Iridovirus, Chloriridovirus, Lymphocystivirus, Megalocytivirus, and Ranavirus[Ranavirus. In past research, ranaviruses received little attention as most infections were deemed subclinical; however, recent ranavirus infections have resulted in considerable morbidity and mortality in a range of wild and cultivated amphibian species in the Americas, Europe, and Asia [Global amphibian populations have declined considerably in recent years, in part due to habitat fragmentation, pollution, and the chytrid fungus robatidis,2. More  capsids , and is Ranavirus. Specifiand Asia -8. It haand Asia , and thaand Asia . Detectiand Asia . Ranavirand Asia .Ranavirus[Frog virus 3 (FV3) is the type species of the genus Ranavirus. FV3′s gRanavirus. DependiRanavirus,13,14. FRanavirus. While oGenetic comparison of related DNA viruses has proven to be an important tool in classifying viral strains and understanding the epidemiology and evolution of different genotypes . By analIn addition to nucleotide insertions/deletions, variation between viral genomes may occur at repeat regions. Eaton et al.  identifiPast studies on the genetic variation between DNA viral strains have allowed for the detection of minute genetic changes that would otherwise have gone unnoticed. These kinds of changes have proved useful in explaining phenotypic differences and evolutionary histories. The purpose of the present investigation is to narrow our focus even further by comparing the genomes of closely related FV3 isolates, including those with varying levels of virulence. This will be done in an attempt to explore the genetic diversity present in strains of FV3, with the ultimate goal of further elucidating the possible genetic basis behind FV3′s unpredictable infectious behaviour.r induced different degrees of infection, L. pipiens tadpoles were exposed to each strain. Tadpoles were exposed to FV3-infected water for 12 h before being transferred to FV3-free water for the remainder of the experiment. The tadpoles were monitored for 41 days, at which time all tadpoles had either died or reached metamorphosis. The results of the survival analysis revealed significant differences in strain effect on tadpole mortality rates . Although deaths triggered by wt-FV3, aza-Cr, and SSME stocks peaked between 15 and 18 days post infection, overall mortality was higher following infection with wt-FV3 and aza-Cr virus (>96%) than with SSME (84%). Wt-FV3 induced a mortality of 97%, and aza-Cr a mortality rate of 96%  , with a In order to investigate the possible genetic variation within coding regions and regions of high variability between closely related viral isolates, we compared our sequenced SSME genome with four published ranavirus genomes. The genomes compared included: SSME, wt-FV3 , rana grUpon analysis of selected ranaviral genomes, a 757 bp deletion was discovered in SSME, deleting the entire 65L coding region and the majority of the 66L coding region Figure . This deAnother coding region demonstrating variation was 50L. An in frame deletion of 13 amino acids was found in 50L when compared to wt-FV3, present only in the SSME genome  to investigate. These regions included: Region 1 ; Region 2 ; and Region 3 , based on the wt-FV3 sequence. We performed a repeat analysis on our sequenced SSME genome that we had previously evaluated for coding region variability, and reference genomes RGV, SSTIV, and TFV. We also decided to analyze the repeat regions of wt-FV3 and aza-C in 1987 .r shared a distinctive repeat sequence, containing a G rather than an A at the 9th nucleotide position , and the nature of comparison , set our investigation apart from past studies. We found that the 3 strains we examined displayed slightly different levels of virulence during r. This variation could be due to the fact that SSME was isolated from a spotted salamander rather than anurans, and so the strain may have evolved in order for it to better adapt to its novel host, as is seen during serial passage [in vivo study in anurans  between the strains, supporting our in vivo finding that the two strains cause essentially the same amount of tadpole mortality (97% and 96% respectively).Our results demonstrate that genetic variation is present between closely related FV3 isolates in both coding and non-coding regions. The SSME genome was sequenced and compared to the published wt-FV3 genome, along with related ranaviral genomes RGV, SSTIV, and TFV. Comparisons revealed that SSME was divergent from wt-FV3, and aza-C passage . For exa passage . This fis Figure . Thus, gOne of the most significant genetic variations found in SSME was a 757 bp deletion that deleted all of the 65L coding region and most of the 66L coding region Figure . Among oSingle nucleotide deletions were found in multiple ranaviruses within the 43R genes Figure  and 2 geThe changes present in 43R, 46L, 49/50L, 65L, and 66L represent the main variable regions amongst the related ranaviral isolates we analyzed. This suggests that these are changeable areas across ranaviruses, and could be used in the future to help explain variable infectious phenotype. Moreover, multiple amino acid deletions present in 65L, 66L, and 49/50L, were limited to the SSME strain, which displayed the lowest level of virulence during tadpole infection. Thus, changes that are unique to SSME may present areas of the genome that are particularly effective in viral attenuation, specifically in an alternative host. As FV3 mortality and morbidity continues to worsen and fluctuate across environmental regions, examination of these genomic areas may prove useful as an initial way to investigate the genetic basis behind infective changes [r to check for repeat number stability across their past viral passages. Analysis revealed that repeat copy number was variable between isolates, even between those from the same geographic location, but that there was some conservation  were isolated from various individuals infected with the virus. The isolates were then tested for variable repeats in 24 polymorphic regions, and based on this analysis, each viral isolate was designated as an individual strain of HCMV . Many ofOther studies have used coding instead of non-coding repeat regions to identify viral strains ,36. In oIn this study we have been able to uncover fine scale genetic variation between closely related ranaviral isolates that have different levels of virulence. We have shown that substantial genetic variability is present between closely related FV3 isolates, both in terms of deletions/insertions, and even more so at select STR locations. These genomic areas with deletions/insertions may present regions that affect viral infectious phenotype, and therefore require investigation. Furthermore, we have identified STR regions that may prove useful in future phylogeographical tracking and identification of ranaviral strains across different environmental regions. As FV3 leads to more unexplained lethal infections in amphibian populations, studies such as this are necessary. The genetic insight that they provide into ranaviral genomes will prove invaluable when seeking to explain the variability of FV3 infectious phenotype in wild populations, and in preventing the devastation that ensues.r, were analyzed in this study and were provided by Professor Gregory Chinchar from the University of Mississipi Medical Center . SSME was isolated from a wild, spotted salamander population in Maine, USA, while aza-Cr was derived from the laboratory wt-FV3 strain through selection with azacytidine [r have been previously sequenced and found to be identical, with wt-FV3′s nucleotide sequence deposited into GenBank [FV3 isolate SSME, wt-FV3, and aza-Ccytidine . Both wt GenBank . AmphibiLithiobates pipiens tadpoles, approximately Gosner stage 25 [r). According to past experiments, such concentration is known to induce sublethal effects in these laboratory conditions [In order to monitor survival of FV3 infected tadpoles, we obtained stage 25 , from thstage 25 . To infenditions . Controlnditions . Startinnditions . Tanks wIn order to check for ranavirus infection, all animals (including euthanized ones) were dissected to remove the liver that was then crushed into a 1.5 ml Eppendorf tube. The resulting tissue mixture was used for DNA extraction. DNA was extracted using QIAmp DNeasy Kit following the standard protocol (Qiagen). After extraction, a double blind PCR was performed using a primer set known to successfully amplify a portion of the major capsid protein within the FV3 genome: MCP-ranavirus-F (5′-GACTTGGCCACTTATGAC-3′) and MCP-ranavirus-R (5′- GTCTCTGGAGAAGAAGAA), following the PCR conditions listed in Mao et al. , using 12 flask, with cells infected at a multiplicity of infection (MOI) of 1 PFU/cell. The cells were harvested 5 days post infection (once cytopathic effect appeared), and viral DNA was extracted using the Purelink Viral RNA/DNA Mini Kit according to manufacturer’s protocol .The SSME viral isolate was propagated on a confluent monolayer of A6 cells, and grown in a 75 cmStandard kits and protocols developed by the manufacturer were used to sequence the SSME sample on a 454 GS-FLX platform (Roche Diagnostics Corporation). Briefly, a Rapid Library Preparation Kit  was used to mechanically shear 500 ng of template DNA into short fragments. A universal sequencing primer that included a short DNA sequence unique to the sample (MID tag) was then annealed to both ends of each DNA fragment. A GS Junior Titanium Emulsion PCR Kit  was used to amplify the sample library, which was sequenced using a GS Junior Titanium Sequencing Kit . In order to assemble a full genomic sequence, the short sequences produced by 454 sequencing were aligned with the reference FV3 genome, wt-FV3, using GS Reference Mapper . Any gaps in the assembled genome were then sequenced using custom PCR primers specific to each gap, with sequencing performed by the Robarts Sequencing Facility . The final genomic sequence was deposited in GenBank accession number KJ175144.FV3 environmental samples were collected from frogs caught by hand at various sites along a lakeshore in Manitoulin, Kagawong, ON, Canada . The frogs were caught using disposable gloves which were changed between each animal inspection. This method is preferred to the ‘net-catching method’ as it has been suggested that cross contamination can occur via the net. Each individual was toe clipped following the protocol #2009-03-04 approved by the Laurentian University Animal Care Committee for tissue sample collection. DNA was then isolated from toe clippings using the DNeasy Blood & Tissue Kit according to the manufacturer’s protocol for total DNA extraction from animal tissues . Samples then underwent PCR with primers designed to amplify specific repeat regions identified in Eaton et al. . Primers2 solution , 5X TAQ DNA polymerase , 10 mM deoxyribonucleotide triphosphates (dNTPs), 0.1 mM primer, 2.5 ng DNA, and water to a final volume of 50 μl. The reactions were then placed in a thermocycler under the following conditions: 94°C for 3 minutes, 94°C for 30 seconds, 56°C for 1 minute, and 72°C for 1 minute for 30 cycles. Sequences of PCR products were determined by Robarts Research Institute DNA Sequencing Facility in London, ON, and were analyzed using BioEdit v7.0.5.In a PCR reaction tube the following reactants were combined: 10X PCR buffer , 50 mM MgClThe authors declare that they have no competing interests.in vitro, carried out the molecular genetic studies, participated in 454 GS-FLX sequence alignment, and drafted the manuscript. SG participated in 454 GS-FLX sequence alignment. PE collected viral isolates from the field, carried out the immunoassays, and participated in the design of the study. DL participated in the design of the immunoassays. CJK oversaw the 454 GS-FLX sequencing. CRB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the final manuscript.EAM participated in the design of the study, isolated viral strains


The female condom is the only evidence-based AIDS prevention technology that has been designed for the female body; yet, most women do not have access to it. This is remarkable since women constitute the majority of all HIV-positive people living in sub-Saharan Africa, and gender inequality is seen as a driving force of the AIDS epidemic. In this study, we analyze how major actors in the AIDS prevention field frame the AIDS problem, in particular the female condom in comparison to other prevention technologies, in their discourse and policy formulations. Our aim is to gain insight into the discursive power mechanisms that underlie the thinking about AIDS prevention and women’s sexual agency.We analyze the AIDS policies of 16 agencies that constitute the most influential actors in the global response to AIDS. Our study unravels the discursive power of these global AIDS policy actors, when promoting and making choices between AIDS prevention technologies. We conducted both a quantitative and qualitative analysis of how the global AIDS epidemic is being addressed by them, in framing the AIDS problem, labelling of different categories of people for targeting AIDS prevention programmes and in gender marking of AIDS prevention technologies.We found that global AIDS policy actors frame the AIDS problem predominantly in the context of gender and reproductive health, rather than that of sexuality and sexual rights. Men’s sexual agency is treated differently from women’s sexual agency. An example of such differentiation and of gender marking is shown by contrasting the framing and labelling of male circumcision as an intervention aimed at the prevention of HIV with that of the female condom.The gender-stereotyped global AIDS policy discourse negates women’s agency in sexuality and their sexual rights. This could be an important factor in limiting the scale-up of female condom programmes and hampering universal access to female condoms. Central in the formulation of global AIDS policies is the creation of access to the existing evidence-based HIV prevention technologies . CondomsGender, which generally denotes the social and cultural constructions of femininity and masculinity, is multi-dimensional and works out differently among different cultural contexts. Feminist theories and women’s health research share the common intention of reflecting critically on biology as a stable and fixed framework. Gender produces diversity. Some consider gender as “a set of practices that bring reproductive distinctions between human bodies into social processes” . It is tGlobal AIDS policies, similar to other policies , result Framing is a necessary, nonetheless often implicit dimension of policy formulation . It is dIn this article, we study how global AIDS policy actors frame the AIDS problem in relation to women’s sexuality and the female condom by analyzing the discourse used in policy papers. It is beyond the scope of this article to prove the impact of this discourse on female condom use. As the female condom is a technology used during sexual intercourse, and as AIDS concerns a sexually transmitted disease that affects people’s sexual and reproductive health, we choose to analyze how the AIDS problem is being framed by global AIDS actors in the context of sexuality. Sexuality is a central aspect of humanity, encompassed by gender ideologies. Both gender and sexuality are culturally constructed and key to consider when addressing the AIDS problem –24.The World Health Organisation (WHO) defines sexual health as a state of physical, emotional, mental and social wellbeing related to sexuality. Sexual health requires a positive and respectful approach to sexuality and sexual relationships, as well as the possibility of having pleasurable and safe sexual experiences, free of coercion, discrimination and violence. For sexual health to be attained and maintained in a population, the sexual rights of all persons must be respected, protected and fulfilled . This apLabelling people by classifying them into target groups with specific characteristics is a common practice in development cooperation, and global AIDS programmes are no exception . AIDS poAs labelling determines access to resources and services, certain population groups organize themselves around a perceived common identity and seek to make themselves visible in society, in particular towards the state, service providers and aid agencies. Groups of people may use an established label to influence global AIDS policy actors. The gay movement in the western world , and theBut labelling has its downsides too. Being pointed out as a high-risk group or victim in global AIDS policies, may lead to stigmatization and exclusion. HIV-positive people often experience stigma, discrimination and blame ,39. And AIDS policy actors are in a position to mark some technologies as more appropriate than others, and to leave certain solutions as unmarked. We are interested in the gender marking of health technologies and the effect this may have on people’s perceptions and acts. The word “condom” for instance is an unmarked term as far as gender is concerned: it is a device that is designed for the male body. As with many other supposedly gender neutral terms, however – think of youth criminals, politicians or house-keepers – most people automatically associate these with one of the two sexes. Yet, by not gender marking a term, this common sense association goes unnoticed. Other words, however, are typically marked in gender terms; for example, the female condom and male circumcision. Gender marking is generally applied to situations or technologies in which the normative and so-called non-normative categories are hierarchically positioned. The gender unmarked sense often remains politically unnoticed, such as “condom,” a term that is often used to designate the male condom. The marked sense, on the other hand, is more heavily articulated and observed .Our study primarily aims to unravel the discursive power of global AIDS policy actors in depicting women’s sexuality and sexual rights and in prioritizing specific AIDS prevention technologies for specific categories of people, in particular the female condom. To assess this power and the way it works out in policy papers of global AIDS actors, we use the concepts of framing, labelling and gender marking. Basic to our approach is seeing policies not only as “data,” but also as “discourse.” “Data” because policies give information on the AIDS problem, population groups and proposed solutions; and “discourse” because policy makers employ a specific terminology and narrative strategies when defining their target groups and stating their objectives, which may in turn affect the persons involved and their attitudes and acceptance of certain prevention technologies . In analWe seek an answer to the question what the discursive power of global AIDS policy actors has been in dealing with women’s sexuality and sexual rights, and in particular the female condom. This central question is divided into three sub-questions. What AIDS prevention technologies do global AIDS policy actors prioritize in their policy papers? How do they frame the AIDS problem in the context of gender and sexuality? How are categories of people labelled, and what are the consequences for the gender marking of AIDS prevention technologies? After presenting our methodology, we describe the results of our analysis in relation to these questions, followed by a discussion and conclusion.This article examines the policies of 16 international AIDS actors that are most essential to the global AIDS governance structure . As noteThe year of appearance of the latest policy paper for each actor varies, but at the time of writing this article, all 16 AIDS policies are valid. On average, an AIDS policy paper consists of 37 pages, ranging from 3 to 68 pages. The purpose of our analysis is not to highlight differences between the agencies, but rather to detect commonalities in the way they prioritize AIDS prevention technologies, how they frame the AIDS problem in the context of gender and sexuality, and consequently the way they label people to target programmes and gender mark their technologies.First, we listed all AIDS prevention technologies that the 16 actors refer to in their policies. We counted a total of nine different technologies and grouped them, as recommended by the Interagency Coalition on AIDS and Development, in the existing and potential technologies . Five exSecond, having identified the nine technologies, we counted the frequency with which each of them was mentioned. The frequency count gives insight into the relative importance and priority accorded to each technology by the 16 actors. From our epistemological perspective, digital frequency counts are important, as they reflect an underlying power to define what is important and at the same time produce certain views and assumptions . As to tThe labels and the frequency with which they are used can inform about global AIDS actors’ assumptions about the target groups of their policies . We therTo add further substance to the discourse on the female condom, we analyzed United Nations Population Fund (UNFPA) experiences on female condoms. UNFPA falls outside our 16 source documents, since it does not have a stand-alone AIDS policy. However, since UNFPA is the designated UN lead agency on SRHR, we analyzed their latest official document on female condoms as an extra source document . An extrThe condom is the most frequently mentioned AIDS prevention technology (30% of more than a thousand times that specific AIDS prevention technologies are mentioned in the 16 source documents). They are mentioned by all but 3 of the 16 actors: the BMGF, the Global Fund and UNESCO. Condoms are in frequency followed by test-and-treat (19%) and PMTCT (11%). Both technologies are based on anti-retroviral treatment and together they represent 30% of the total number of times a technology is mentioned. Male circumcision comes fourth: it is mentioned by 12 agencies with a total frequency of 12%. Sexuality education is mentioned by seven agencies, with a total frequency of 11%, mainly in the policy papers of UNESCO and UNICEF. The majority of agencies (9 out of 16) do not mention sexuality education in their policies. The three technologies that are still in their trial phase are mentioned for a total of 12%, including microbicides (5%), vaccines (4%), and PrEP (3%).AIDS, AIDS and Behavior, and AIDS Patient Care and STDs; all articles published before July 2012. These 10 articles actually considered male condoms only when mentioning condoms. When articles do consider female condoms, they specifically mark them. This extra analysis demonstrates that female condoms are indeed generally excluded when the gender unmarked technology of condoms is addressed. This means that we can safely assume that when the gender unmarked term condom is used, it typically involves the male condom, and when female condoms are involved they are explicitly mentioned.Although the condom is mentioned by 13 actors with a total frequency of 30%, only nine agencies mention the female condom with a total frequency of 4%, most of which is accounted for by PEPFAR. Besides BMGF, who missed out on the condom, an additional six global AIDS policy actors  that mentioned the condom completely miss out on the female condom. It might be possible that some agencies group the female condom under the gender unmarked technology “condom.” To find out if people, and thus also likely global AIDS policies, refer to both male and female condoms when using the generic term condom, we studied 10 most recently published articles with condom(s) in the title in three journals: The limited attention to female condoms as one of the AIDS prevention technologies stands in sharp contrast to the multiple references made to other technologies. Two technologies, microbicides and vaccines, are still in the development stage and therefore still not proven effective to be widely implemented . They arWe conclude that some technologies of which the effectiveness is partial, such as male circumcision and microbicides, catch more attention in the number of times mentioned in the analyzed text  than the female condom (4%), which is proven effective. This suggests a bias in policy preference which is not supported by the available evidence on the efficacy of available technologies. The high recognition of the male condom, as an effective unmarked technology, compared to the low recognition of the female condom, as a marked technology, is noteworthy and solicits the question how the problem of AIDS is framed in the context of gender and sexuality.Of all four terms considered, the term “gender” is by far most often mentioned (53%). All 16 AIDS policies, except the one of the BMGF, somehow give importance to gender as illustrated by the following quotes. The UNDP policy paper states for example: “HIV/AIDS is not only about a virus. It is also about shame and guilt, gender inequality, power relations, silence and denial, stigma and discrimination” , p. 4. TReproductive health (23%) is used more often than sexuality (18%). Global AIDS policy actors appear to favour the term gender instead of sexuality, when framing the AIDS problem. Sometimes they are connected. This is illustrated in the DFID policy paper: “Women and men face different risks and barriers in relation to the AIDS epidemic and in accessing services. Gender inequalities mean that women and girls cannot always decide if, when, how and with whom they have sex, or when to access basic services. Violence against women and girls significantly increases their risk of HIV infection. Women and girls report increased violence for refusing sex, requesting condom use, accessing HIV counselling and testing, and for testing HIV-positive. Women and girls also bear the greatest burden of care, including caring for orphans and those who are sick” , p. 24. We should assure women’s ability to control their own fertility. These rights rest on the recognition of the basic right of all couples and individuals to decide freely and responsibly the number, spacing and timing of their children and to have the information and means to do so, and the right to attain the highest standard of sexual and reproductive health. These seven actors proficiently use the discourse of SRHR in their AIDS policies. Especially the Europe based agencies see it as essential. The DFID policy, for example, states: “Expanded access to Sexual Reproductive Health and Rights (SRHR), including family planning, is a key part of an effective AIDS response. Successful HIV prevention is about enabling individuals, couples and communities to make healthy choices about personal aspects of their lives – particularly sexual behaviour” [The term sexual rights is used even less (7%). Apart from the four actors who do not refer to sexuality in their policy papers, an additional five agencies omit the term “sexual rights”: UNESCO, UNIFEM, WB, PEPFAR and GHPWG. Seven agencies, predominantly the ones based in Europe, however, do use the term “sexual rights”: UNAIDS, WHO, UNICEF, EU, Great Britain, the Netherlands and Sweden. In this way, they refer to what was expressed during the ICPD, held in 1994 and re-enforced in 1999 during the 21st special session of the General Assembly of the United Nations:haviour” , p. 15. haviour” , p. 10. We conclude that global AIDS policy actors frame AIDS predominantly in relation to gender and/or reproductive health, rather than to sexuality and sexual rights. Below we will show what this implies for the way people are labelled in AIDS policies.The 16 global AIDS policies use 14 different labels to differentiate people, which altogether were mentioned a total of 2469 times (=100%). Women (27%) are the most frequently mentioned category, much more often than men (7%). Girls (9%) get more attention than boys (2%), and mothers (5%) more than fathers (0.3%). For males, the label homosexual  is used almost as often as that of men (7%), while for females, the label homosexual  is hardly applied. It is mentioned by only two agencies, just once by each.Of the 14 categories, the AIDS policies of nine agencies label five categories of people to target AIDS prevention programmes: youth (25%), sex workers (7%), drug users (7%), gays/men who have sex with men (6%) and mothers (5%) ,57–61,66AIDS policies also categorize people as women (27%), girls (9%), men (7%), boys (2%), fathers (0.3%) and lesbians (0.1%), but these labels are not used for targeting purposes in AIDS prevention programmes , p. 9. WThe discourse on men and boys differs from the one on women and girls. Men are described as highly sexually active, not wanting to use condoms and not taking responsibility for their sexual acts, such as stated in the PEPFAR policy paper: “Males are resistant to the use of condoms” , p. 18. While AIDS policies do differentiate between the sexes by labelling women and men, the category of young people is predominantly left gender unmarked in AIDS prevention. For example, the UNGASS document declares that “young people are leading the global prevention revolution” , p. 5 , We conclude that labelling is a common practice in global AIDS policies in order to target specific population groups. On the one hand it leads to exclusion of certain categories of people from AIDS prevention programmes and technologies; on the other hand it focuses the attention to other groups, with the inherent risk of stigmatizing them. Moreover, the labels that are being used imply different notions of sexual agency for specific groups of people. Before we draw further conclusions, we investigate gender labelling more in-depth, by considering two typically gender marked AIDS prevention technologies more closely: the female condom and male circumcision.Male circumcision (12%) is mentioned three times more often as a prevention technology in global AIDS policies than the female condom 4%; see . Twelve Meanwhile, female condom programmes receive much less support. The global AIDS policies are generally positive about the promotion of condoms, both male and female condoms. The WHO policy document states: “HIV programmes should promote equity between the sexes in sexual decision-making, including negotiation of safer sex and use of male and female condoms” , p. 28. According to UNFPA programming for female condoms has the goal “to develop strategies and programmes through which every sexually active person at risk of HIV or other sexually transmitted infections – regardless of age, marital status, gender, sexual orientation, economic status, cultural and religious beliefs or HIV status – has access to good quality condoms when and where he or she needs them; is motivated to use male or female condoms, as appropriate; and has the information and knowledge to use them consistently and correctly” , p. 6. ITo sum up, the discursive power of global AIDS policy actors in framing the AIDS problem is located in defining gender from the perspective of women and girls, rather than men, and with a focus on reproductive health, rather than sexual health or sexual rights. This implies that women and girls are mainly portrayed as victims, rather than sexual agents. Women and girls are considered as homogenous groups, without any distinction for specific programmes or technologies. Policy makers target women as mothers in PMTCT programmes to prevent their new-borns from becoming HIV infected. Seldom, women are labelled as lesbians, and as a result they are excluded from AIDS policies. This denial of other sexual orientations of women illustrates the general disregard for women’s sexual agency. And this colours the prejudices about the female condom as a prevention technology. We argue that global AIDS policy actors contribute to re-enforcing the denial of women’s sexual agency. Other researchers also found that the existing gender stereotypes about sexual behaviours have hindered AIDS prevention ,86.The social environment represents social and sexual (risk) relations between all kinds of people. Running risks is considered as an expression of tension and of power relations in society. This view on risk means that the whole community is involved and everyone contributes one way or the other to risk-taking behaviour, not only high-risk groups defined as such by others . This apNormalizing female condom use for commercial sex workers rather than for other categories of women who are sexually active, implicitly links HIV to sexually immoral behaviour, thus stigmatizing HIV-positive women in their local communities. Once a woman is known to be HIV-positive, she is often seen as sexually immoral, which in fact is a negative interpretation of her sexual agency ,40. ThisThe nature of the discursive power of global AIDS policy actors was the central theme of this article, with particular reference to the female condom. We found that global policy actors frame the AIDS epidemic mainly in the context of gender and reproductive health, rather than that of sexuality and sexual rights. Women and girls are often referred to, but more as victims than as specific target groups, and not as sexual agents. Female target groups are mostly mothers (focusing on their reproductive role) and commercial sex workers , leaving out women in their own right, as sexual agents. Homosexual men (gays) are explicitly labelled to target AIDS prevention programmes, while homosexual women (lesbians) are not labelled at all. Among the prevention technologies, the condom is prioritized in the policy discourse. Although it remains gender unmarked, it is implicitly associated with men (men only), reinforcing the gender notion of sexual agency of men. It is almost taken for granted that  condoms need to be accessible for all men and not only specific categories of men. In contrast, the female condom is normalized for sex workers. None of the 16 policy papers analyzed make a serious attempt to insist on a programme of action to make female condoms universally accessible, that is: to all sexually active women. There is a deliberate effort to portray male circumcision as the norm for all men in sub-Saharan African countries that have high HIV prevalence rates, but without proper additional sexual education this may create extra risks for female partners. The gender-stereotyped AIDS policy discourse at the global level negates women’s agency in sexuality and her sexual rights. This in turn might have limited the scale-up of programmes that would make female condoms universally accessible.