Physics phenomena of multi-soliton complexes have enriched the life of dissipative solitons in fiber lasers. By developing a birefringence-enhanced fiber laser, we report the first experimental observation of group-velocity-locked vector soliton (GVLVS) molecules. The birefringence-enhanced fiber laser facilitates the generation of GVLVSs, where the two orthogonally polarized components are coupled together to form a multi-soliton complex. Moreover, the interaction of repulsive and attractive forces between multiple pulses binds the particle-like GVLVSs together in time domain to further form compound multi-soliton complexes, namely GVLVS molecules. By adopting the polarization-resolved measurement, we show that the two orthogonally polarized components of the GVLVS molecules are both soliton molecules supported by the strongly modulated spectral fringes and the double-humped intensity profiles. Additionally, GVLVS molecules with various soliton separations are also observed by adjusting the pump power and the polarization controller. Different from the conservative solitons purely originating from the simple balance between dispersion and nonlinearity, continuous flow of energy gets involved in dissipative systems and the composite balance among cavity dispersion, fiber nonlinearity, laser gain and loss of the fiber lasers, plays a significant role in the formation of DSs. Due to the peak power clamping effect, fundamental solitons tend to be unstable after amplification4, and more complex waveforms such as pulse bunching5, harmonic mode-locking7, soliton molecules9 and soliton rain11 can appear with increasing pump power. In particular, soliton molecules (i.e. bound states of solitons), featuring multi-soliton structures, have been a topic of intensive research in fiber lasers. In analogy with the physics of matter, soliton molecules as a multi-soliton complex essentially stem from the interaction of repulsive and attractive forces between particle-like solitons; the formation is a dissipative process that requires the supply of binding energy12. Meanwhile, quite different from the well-known high-order solitons, the soliton molecules will not decay under infinitesimal perturbation2, which ensures their promising applications in optical communications for coding and transmission of information in high-level modulation formats, and increasing capacity of communication channels beyond binary coding limits15. From the first theoretical study of soliton molecules by Malomed16 and Akhmediev et al.17, numerous experimental observations have been reported in nonlinear polarization rotation18, molybdenum disulfide19, carbon nanotube20, and figure-of-eight21 based fiber lasers. In particular, tightly bound states are the most conventional type, which are interpreted in terms of fixed and discrete soliton separations. Additionally, various types of soliton molecules, such as vibrating soliton molecules, oscillating soliton molecules22, soliton molecules with flipping and independently evolving phase24, have been also theoretically predicted and experimentally observed.Passively mode-locked fiber lasers have attracted intensive research for the applications of ultrashort pulsed light source with unprecedented pulse energy or repetition rate, as well as the exploration of dynamics of optical dissipative solitons (DSs)27. S. T. Cundiff et al. first demonstrated that both the group-velocity-locked vector solitons (GVLVSs) with rotating polarization state and the phase-locked vector solitons (PLVSs) with fixed polarization state could be formed in fiber lasers29. L. M. Zhao et al. further revealed that with moderate linear cavity birefringence, another type of GVLVSs with a unique feature of two sets of Kelly sidebands could be formed in fiber lasers30. In particular, the two orthogonally polarized components of such vector solitons shift their central frequency in opposite directions to compensate the birefringence-induced group-velocity difference. Thereupon, these two components can trap each other and travel as a unit in time domain. Especially, the two set of Kelly sidebands are interpreted as the indicator of the central frequency shift; and the stronger birefringence is the laser cavity, the larger wavelength shift of the spectrum. In addition, vector solitons with locked and precessing states of polarization32, vector solitons in dispersion-managed regime33 and high-order PLVSs34 have been reported in fiber lasers. More recently, the mergence of soliton molecules and vectorial nature of light spreads a new concept of vector soliton molecules that inspires us to reveal more interesting behaviors and underlying dynamics of these compound multi-soliton complexes formed at both time domain and polarization directions in a unique testbed37. It is found that vector solitons as a non-dispersive unit can also attract or repel each other to form the vector soliton molecules, which provide a possibility for twofold increasing the optical communication capacity.If no polarization discrimination component is used in fiber lasers, the two orthogonally polarized components could be coupled together to form another type of multi-soliton complexes, namely vector solitons in fiber lasers, which not only possess much more plentiful behaviors and richer dynamics than their scalar counterparts, but also pave a promising way for numerous applications from nano-optics to high-capacity fiber optic communicationsIn this paper, we report an experimental manifestation of GVLVS molecules in a birefringence-enhanced fiber laser. With enhanced net cavity birefringence introduced by a segment of polarization-maintaining fiber (PMF), GVLVSs interpreted as a multi-soliton complex are formed. The two orthogonally polarized components of the vector solitons possess obvious central wavelength shift and travel as a non-dispersive unit in the laser cavity at the same group velocity. Furthermore, with appropriate settings, GVLVS molecules as compound multi-soliton complexes are obtained. Polarization-resolved measurement is adopted to explore the vectorial nature of the GVLVS molecules. In addition, GVLVS molecules with various soliton separations are experimentally observed. The proposed fiber laser serves as an ideal testbed for both enriching the DS community and extending their potential applications.The experimental setup is schematically depicted in Fig. 2 pulse shape. The time-bandwidth product is about 0.354, indicating that the output pulse is nearly transformed-limited. By decreasing the pump power to 38 mW, the fiber laser approaches to the fundamental mode-locking regime with an output power of 65 μW, corresponding to the pulse energy of 6.8 pJ. Although the pump power is less than the threshold, mode-locking operation can be still maintained due to the pumping hysteresis phenomenon. Figure With appropriate settings, self-started mode-locking of the fiber laser is achieved by simply increasing the pump power above the mode-locking threshold of about 47 mW, and the mode-locked pulses are automatically evolved into vector solitons as no polarization-sensitive components are adopted in the laser cavity. We increase the pump power to 300 mW and obtain a stable mode-locking state with an output power of 4.5 mW. The enhanced net birefringence of the laser cavity leads to the formation of GVLVSs as illustrated in Fig. From fundamental operation regime, through increasing the pump power, the fiber laser tends to operate at multi-pulse states due to the soliton energy quantization effect. In particular, the dual- and triple-pulse states are respectively observed with pump power of about 41 mW and 45 mW as shown in Fig. 2 pulse shape is assumed; the soliton separation is estimated to be 2.3 ps, exactly matching the modulation period of the spectral fringes. The soliton separation is approximately 2.8 times of the pulse width, and the height ratio of the three peaks of the autocorrelation trace is around 1:2:1. Thus it can be seen that two GVLVSs of identical intensity are tightly bound together to form the GVLVS molecule. Figure By carefully manipulating the intra-cavity polarization state, these separate particle-like solitons can be bound together to form soliton molecules due to internal interaction, which are interpreted as another type of multi-soliton complexes formed in time domain. As shown in Fig. 30 is adopted to get insight of the vectorial nature of these GVLVS molecules. Figure 2 pulse shape, and soliton separation of 2.3 ps. Compared to the pulse width of the individual soliton presented in Fig. Furthermore, polarization-resolved measurement2 pulse shape is assumed. Thus it can be seen that the two GVLVSs are tightly bound together38. Additionally, Fig. The adjustment of the pump power and the intra-cavity PC leads to the change of the GVLVSs, which further plays a role in the soliton interaction of repulsive and attractive forces. Hereby, GVLVS molecules with various soliton separations are also observed in this fiber laser. Particularly, GVLVS molecules shown in Fig. We develop a birefringence-enhanced fiber laser to explore the new concept of GVLVS molecules. As no explicit polarization sensitive components are adopted and fibers are weakly birefringent in the laser cavity, the formed solitons are essentially vector solitons. The PMF is purposefully utilized in the cavity thus local strong birefringence is introduced. Therefore, the orthogonally polarized solitons experience fast polarization evolution, which facilitates the formation of GVLVSs. Meanwhile, we explore the dynamics evolution of GVLVSs depending on different birefringence. In particular, different-length PMFs are respectively spliced inside the cavity, thus introducing the birefringence-management technique. Apart from 15 cm PMF, Fig. Next with careful polarization-state manipulation, these separate particle-like GVLVSs can be bound together at time domain to form the compound multi-soliton complexes, namely GVLVS molecules. The GVLVS molecules behave themselves with dual properties. On one hand, the unique feature of soliton molecules is characterized by the strongly modulated spectral fringes, and further verified by the double-humped intensity profile. On the other hand, the GVLVS molecules display distinct spectrum of two set of Kelly sidebands, and their two orthogonally polarized components are both soliton molecules with the identical soliton separation. Besides, by finely adjusting the pump power and intra-cavity polarization state, GVLVS molecules with various soliton separations could be experimentally observed. Overall, the developed birefringence-enhanced fiber laser approaches to the operation regime of GVLVS molecules. The investigation on the interesting behaviors and underlying dynamics of these compound multi-soliton complexes provides the opportunities for both enriching the life of DSs and extending their potential applications.An optical spectrum analyzer  with a resolution of 0.02 nm and a 1 GHz real-time oscilloscope  together with a 2 GHz photodetector  are respectively employed to monitor the optical spectra and the pulse train of DSs. RF spectrum is analyzed by a 3 GHz electrical spectrum analyzer . Finally, the pulse profile is measured by a commercial autocorrelator (Femtochrome FR-103HS).


Tuberculosis (TB) in children is frequently paucibacillary and non-severe forms of pulmonary TB are common. Evidence for tuberculosis treatment in children is largely extrapolated from adult studies. Trials in adults with smear-negative tuberculosis suggest that treatment can be effectively shortened from 6 to 4 months. New paediatric, fixed-dose combination anti-tuberculosis treatments have recently been introduced in many countries, making the implementation of World Health Organisation (WHO)-revised dosing recommendations feasible. The safety and efficacy of these higher drug doses has not been systematically assessed in large studies in children, and the pharmacokinetics across children representing the range of weights and ages should be confirmed.SHINE is a multicentre, open-label, parallel-group, non-inferiority, randomised controlled, two-arm trial comparing a 4-month vs the standard 6-month regimen using revised WHO paediatric anti-tuberculosis drug doses. We aim to recruit 1200 African and Indian children aged below 16 years with non-severe TB, with or without HIV infection. The primary efficacy and safety endpoints are TB disease-free survival 72 weeks post randomisation and grade 3 or 4 adverse events. Nested pharmacokinetic studies will evaluate anti-tuberculosis drug concentrations, providing model-based predictions for optimal dosing, and measure antiretroviral exposures in order to describe the drug-drug interactions in a subset of HIV-infected children. Socioeconomic analyses will evaluate the cost-effectiveness of the intervention and social science studies will further explore the acceptability and palatability of these new paediatric drug formulations.Although recent trials of TB treatment-shortening in adults with sputum-positivity have not been successful, the question has never been addressed in children, who have mainly paucibacillary, non-severe smear-negative disease. SHINE should inform whether treatment-shortening of drug-susceptible TB in children, regardless of HIV status, is efficacious and safe. The trial will also fill existing gaps in knowledge on dosing and acceptability of new anti-tuberculosis formulations and commonly used HIV drugs in settings with a high burden of TB. A positive result from this trial could simplify and shorten treatment, improve adherence and be cost-saving for many children with TB.Recruitment to the SHINE trial begun in July 2016; results are expected in 2020.ISRCTN63579542, 14 October 2014.International Standard Randomised Controlled Trials Number: PACTR201505001141379, 14 May 2015.Pan African Clinical Trials Registry Number: Clinical Trial Registry-India, registration number: CTRI/2017/07/009119, 27 July 2017.The online version of this article (10.1186/s13063-018-2608-5) contains supplementary material, which is available to authorized users. Of the estimated 10.4 million new tuberculosis (TB) cases globally per annum, approximately one million occur in children in Africa and South East Asia –4. FurthHistorically, studies of anti-tuberculosis treatment in children have lagged substantially behind adult studies and data regarding TB treatment extrapolated from adults. Children have also been seen to pose little risk of onward transmission. In combination with the challenges in confirming a TB diagnosis in children and the resource constraints, the focus has been on adult trials. The approaches used to treat childhood TB have relied on extrapolation to children of the evidence from clinical trials in adults who have a different spectrum of TB disease and lower bacillary burden .The TB disease spectrum observed in children is different compared to adults , 10. WheMycobacterium tuberculosis exposure .Tab.Tab54].TThe investigational medicinal products in this trial will be rifampicin, isoniazid, pyrazinamide and ethambutol. ARs include any untoward or unintended response to any of these drugs.All AEs meeting the standard definitions above are reported, regardless of their relationship to TB. The seriousness of the events is assessed using the ICH-GCP definitions, and severity is determined in accordance with the 2014 Division of AIDS toxicity grading scale . The cauAll SAEs, grade 3/4 adverse reactions and notable events  are notified to the Medical Research Council Clinical Trials Unit (MRC CTU) within one working day of the investigator becoming aware of the event. Other reportable events are reported within seven working days. The investigators at each site are also responsible for the onward reporting of adverse events to their ethics and regulatory bodies, as per their local reporting requirements. MRC CTU is responsible for annual onward reporting of a line listing of events to the trial sponsor, University College London (UCL). Investigators are responsible for reporting all SAEs, notable events and other adverse events meeting their reporting requirements that occur from the time of randomisation until 30 days after the last protocol treatment administration.UCL is responsible for trial oversight and ensuring that arrangements are in place for adequate management, monitoring, analysis and reporting of the trial.Quality assurance and quality control are based on a formal risk assessment which leads to the development of the trial quality management plan implemented by the sponsor. On-site monitoring and data validation and assessment of adherence to International Conference on Harmonisation-Good Clinical Practice (ICH-GCP) principles is regularly conducted by independent local monitors at each site and periodically directly by MRC CTU on behalf of the sponsor. Each site is responsible for its own data entry and local trial management.The trial is overseen by a number of committees each with distinct functions and membership. The Trial Steering Committee (TSC) consists of the chief investigator, site principal investigators and independent members. The TSC provides overall supervision for the trial and advice through its independent chair. The ultimate decision for the continuation of the trial lies with the TSC.The Independent Data Monitoring Committee (IDMC) reviews confidential, accumulating data for the trial produced by the trial statisticians. The functions and frequency of its meeting are dictated by the DMC charter. The IDMC can recommend premature closure or reporting of the trial.The Endpoint Review Committee (ERC), blinded to the trial arm, will determine the primary endpoint classification for all participants as favourable, unfavourable or unassessable. In addition, the ERC will examine evidence and adjudicate causes of death based on all available sources of data and determine the TB diagnosis at enrolment across sites for the key secondary analysis of those with definitive TB .SHINE is the first large-scale paediatric trial that will test whether 4-month therapy is as effective as the standard 6-month course in children with non-severe TB. Recently, three large multicentre trials to determine whether fluoroquinolone-containing regimens can shorten treatment from 6 to 4 months did not demonstrate non-inferiority of the 4-month regimens in adults with drug susceptible TB –46. In STB studies in children have long been neglected owing to the perception that paediatric TB is not a public health priority. Paediatric TB trials, in particular, face challenges because of difficulties in confirming TB bacteriological conversion as a study endpoint . While aThe trial includes HIV-infected children as current evidence suggests no association between treatment duration and outcomes in patients on ART receiving rifampicin-containing regimens . AccordiEconomic analyses will assess the cost-effectiveness of reducing treatment from 6 months to 4 months while the social science analyses will evaluate the palatability and acceptability of using the recently implemented paediatric anti-tuberculosis FDCs.SHINE will confirm whether or not treatment-shortening of drug susceptible TB in children is efficacious and safe. It will also fill existing gaps in knowledge on dosing of the new anti-TB formulations and commonly used HIV drugs in settings with a high burden of childhood TB. A positive result from this trial (i.e. if 4 months is shown to be non-inferior to 6 months) will be to simplify treatment, to potentially improve adherence and to reduce treatment costs for a large proportion of children with TB


Evidence on contraceptive self-injection from the United States and similar settings is promising, and the practice may increase access. There are no published studies on the feasibility of contraceptive self-injection in sub-Saharan Africa to date. The purpose of this study was to assess feasibility of subcutaneous depot medroxyprogesterone acetate self-injection in Uganda, with specific objectives to (a) measure the proportion of participants who self-injected competently, (b) measure the proportion who self-injected on time 3 months after training (defined conservatively as within 7 days of their reinjection date) and (c) assess acceptability.In this prospective cohort study, 380 18–45-year-old participants completed self-injection training by licensed study nurses, guided by a client instruction booklet, and practiced injection on prosthetics until achieving competence. Nurses supervised participants' self-injection and evaluated injection technique using an observation checklist. Those judged competent were given a Sayana® Press unit, instruction booklet and reinjection calendar for self-injection at home 3 months later. Participants completed an interview before and after self-injection. Nurses visited participants at home following reinjection dates; during the follow-up visit, participants demonstrated self-injection on a prosthetic, injection technique was reevaluated, and a postreinjection interview was completed.Of 368 participants followed up 3 months posttraining, 88% [95% confidence interval (CI) = 84–91] demonstrated injection competence, and 95% (95% CI=92–97) reinjected on time, while 87% (95% CI=84–90) were both on time and competent. Nearly all (98%) expressed a desire to continue.Self-injection is feasible and highly acceptable among most study participants in Uganda.The first research results on contraceptive self-injection in sub-Saharan Africa indicate initial feasibility and acceptability of the practice 3 months after women received one-on-one training and a highly visual training and memory aid. Results can inform self-injection programs which aim to increase women's autonomy and access to injectable contraception. DMPA is effective and safe in either form. A recent review of 14 studies concluded that DMPA-SC and DMPA-IM are therapeutically equivalent, with similar side effects profiles Sayana® PressAlthough self-injection of contraceptives is relatively new, a limited number of studies have been conducted. Pfizer Inc. tested the safety and efficacy of self-injection of DMPA-SC in a prefilled syringe involving 6279 women-cycles of exposure via self-injection The purpose of our study was to assess feasibility and acceptability of self-injection with DMPA-SC in Uniject™ in Uganda. The two primary objectives were to measure the proportion of study participants who demonstrate competent self-injection technique 3 months after training and to measure the proportion who self-inject on time. Our secondary objective was to assess the acceptability of self-injection reflected in the proportions who expressed a desire to continue with self-injection and who reported that they were likely or very likely to recommend the practice to others.22.1This prospective cohort study was conducted in three public facilities in Mubende District and one NGO clinic and two public facilities in Gulu District of Uganda from June to December 2015. These sites included two rural facilities, two urban hospitals, an urban facility that serves youth and does outreach to rural areas and a periurban facility located near a college. Female participants were 18 to 45 years of age and eligible to receive injectable contraception per standard of care guidelines. All had sought care at the clinic and decided to use injectable contraception prior to recruitment, and all provided signed informed consent. Anyone who was pregnant, did not reside permanently in the area, felt unwell on the day of enrollment or did not speak the primary language of the area was excluded from the study. The study received institutional review board (IRB) approval from the PATH Research Ethics Committee and the Mulago Hospital IRB in Uganda.Study procedures were implemented by licensed nurses who had been trained in Sayana® Press administration, research ethics , interviewing, and how to train and counsel women for self-injection.Study participants were trained one-on-one by study nurses guided by a client instruction booklet (Appendix A) At enrollment and after training, women self-injected under the supervision of a nurse. Their injection technique was evaluated using an observation checklist (Appendix B) At follow-up, each self-injecting participant was visited at home by the study nurse at least 7 days following her scheduled reinjection date so that it was feasible to assess whether she had completed her injection on time. The participant demonstrated the injection steps on a prosthetic device without guidance from the study nurse, and her technique was evaluated according to the observation checklist. If she had forgotten to do the injection, the nurse observed and evaluated as she gave herself the injection. If she did not want to self-inject but wished to continue with the method, the nurse administered the injection. Every woman completed a follow-up interview, regardless of whether she self-injected.2.2Data were collected via structured questionnaires in private face-to-face interviews with each study participant. Interviews were conducted in the local language  by female interviewers, and data were entered electronically on Android tablets. The same study nurses who enrolled women conducted the follow-up visits.A sample size of 380 self-injectors was calculated to achieve a desired precision of 5 percentage points around the point estimate of 80% of women competent in self-injection at 3 months [95% confidence interval (CI)=75%–85%] and a precision of 5 percentage points around the point estimate of 80% of women reinjecting consistent with the schedule. The sample size assumed 10% lost to follow-up and 20% discontinuation of the injectable.The primary objective of evaluating competency in self-injection had two end points: the percent of women who demonstrated correct self-injection technique consistent with the observation checklist and the percent who reinjected on schedule, i.e., within 1 week of their reinjection date. To qualify as competent, a participant had to successfully demonstrate 5 critical injection steps from the 19-step observation checklist (Appendix B) Data analysis was performed using STATA version 14.1. 95% CIs for the primary end points were calculated using exact CIs for binomial proportions.33.1A total of 384 participants enrolled in the study. Two women enrolled were later determined ineligible due to pregnancy (both screening failures at the time of recruitment). Data were lost for one enrolled participant, and one participant did not complete enrollment study procedures. For baseline data analysis purposes, participants consisted of 380 healthy female adults who agreed to use injectable contraception and were willing to try self-injection. 3.2Among the 380 self-injectors, 98% demonstrated competence immediately after training. Seven women were lost to follow-up, and five women discontinued the injectable for reasons unrelated to self-injection — leaving 368 participants for follow-up analysis. Among these participants, 88% demonstrated competence immediately and 3 months later .iii AmonWith respect to subjective competency, 92% reported that their second injection was “very easy” to do , an incr3.3Nearly all of those who gave two injections (98%) expressed a desire to continue with self-injection (data not shown), and 88% reported they were “very likely” to recommend the practice to others .3.4There were no serious adverse events. The most common adverse events were known side effects of DMPA: amenorrhea and other changes in menstrual bleeding patterns, including spotting. Twenty-two women experienced an injection-site reaction (ISR) in the form of a dimple, bruise, blister or nodule, and three women sought treatment or advice for an ISR. All adverse events were mild to moderate in severity.3.5Nearly all women (98%) felt that they were able to keep the device secure (safe from discovery by children or others) until it was time for reinjection, with most (61%) storing it in a handbag . Discard4This study provides the first evidence from sub-Saharan Africa that self-injection of DMPA-SC in Uniject™ is feasible for the vast majority of women after a single one-on-one training session — including women with limited education and younger women. These results echo previous studies on self-injection from developed countries but further demonstrate that self-injection is feasible and desirable in settings where women have relatively less education and access to health information and services.The results of this study also point to important program and policy implications for potential introduction of injectable contraceptive self-injection Our results provide additional context following the recent recommendation of the World Health Organization that self-injection be offered “in contexts where mechanisms to provide the woman with appropriate information and training exist, referral linkages to a healthcare provider are strong, and where monitoring and follow-up can be ensured” Disposal of used devices is another unresolved challenge. Latrine disposal removes the spent device from risk of contact quickly but may not be an ideal strategy given limited global access to sanitation facilities 4.1By design, self-injection was not directly observed at the 3-month reinjection time, and it is possible that injections demonstrated on the model may not mirror women's actual practices. Moreover, the five women who self-injected at follow-up rather than injecting on a prosthetic reflect a difference in instrumentation of unknown significance. The small number of cases that differ in this regard is unlikely to influence the results. Because of the timing of the follow-up visit (within the 4-week reinjection window for DMPA) An additional limitation is that we did not vary our approach to training clients to self-inject, so it is not possible to assess which specific components of our approach were critical to competent and on-time client reinjection or acceptability — in other words, whether one-on-one training, practice injections or client instruction booklets were critical to our outcomes.Participants were recruited at public-sector family planning clinics from among clients who had selected injectable contraception. Had there been awareness raising regarding the opportunity to try self-injection, a different family planning clientele and possibly more new users may have been attracted. Feasibility and/or acceptability might vary for clients who typically receive contraceptives from community health workers.


We suggest a novel method that uses artificial blood cells  as photosensitizers in dye laser treatment (at 595-nm wavelength) for port-wine stains  based on the results of animal experiments. As compared with human red blood cells, Hb-Vs have the same absorbance of 595 nm wavelength light and produce the same level of heat following dye laser irradiation. Small sized Hb-Vs (250 nm) distribute in the plasma phase in blood and tend to flow in the marginal zone of microvessels. Intravenous injections of Hb-Vs caused the dilatation of microvessels, and dye laser treatment with Hb-Vs destroyed the vessel wall effectively. Following the intravenous injection of Hb-Vs, the microvessels contained more Hb that absorbed laser photons and produced heat. This extra Hb tended to flow near the endothelial cells, which were the target of the laser treatment. These attributes of Hb-Vs will potentially contribute to enhancing the efficacy of dye laser treatment for port-wine stains. Hemoglobin is a type of porphyrin. Thus, our proposed treatment may have aspects of photodynamic therapy using porphyrin that leads to a cytotoxicity effect by active oxygen. Artificial red blood cells have been developed as an alternative to blood transfusions in order to maintain systemic circulation and oxygenation during acute hemorrhage. We suggest a change of this recognition of the sophisticated hemoglobin vesicle (Hb-V) from oxygen carriers to photosensitizers in vessels such as photodynamic therapy. We investigated the use of Hb-Vs as photosensitizers in the dye laser treatment of port-wine stains (PWSs). We summarize our results of in vivo and in vitro experiments to measure the aptitude of Hb-V as a photosensitizer, and discuss the advantages of this method ,2,3.Some birthmarks have the appearance of the skin having been discolored by wine, hence the name “port-wine stain” PWSs are common capillary malformations characterized on histological examination by dysfunctional dilated microvessels in the normal dermis . Their iSince the 1980s, the flashlamp-pumped pulsed dye laser (PDL) has been considered the best laser system for the treatment of PWSs  5,7].,7.5,7].Laser treatment for PWSs is based on selective photothermolysis, in which vascular-selective laser light is absorbed by the hemoglobin (Hb) in red blood cells (RBCs) (the absorbers and heaters); this produces heat, which results in photocoagulation and the aggregation of RBCs and ultimately in necrosis of the endothelial cells (the targets)  7,8,9].,8,9.7,8,Various vascular-selective lasers and light illumination can be used, depending on the condition and position of the affected skin and vessels. These include pulsed dye lasers , AlexandThe aim of these treatments is to change the skin color of the lesion to that of healthy skin by destroying the affected microvessels in the dermis without injuring the surrounding tissue. Such treatments can reduce the severity of complications, including cutaneous hypertrophy, disfigurement of the normal tissue architecture, and psychosocial morbidity ,7. The uDespite the improvements, only one-fifth of PWSs clear completely, although the color of most capillary malformations improves significantly with treatment . In our These laser treatment-resistant PWSs show specific histological characteristics. Some vessels may be too large or the blood flow may be too fast, thus preventing the generation of sufficient energy to damage the vessels irreversibly . It is lIn Japan, dye lasers have been used in trial treatments for PWSs. Before the laser irradiation, the redness of the affected skin was emphasized by prior treatment using methods such as infrared ray heating or cupping therapy , in whicHb-Vs are cellular particles that encapsulate a purified and concentrated Hb solution within a phospholipid bilayer membrane, acting as artificial red blood cells. The final Hb concentration was adjusted to 10 g/dL ,15,16. TThe microvessels of a rat iris are visible with a magnifying glass through the transparent cornea; this allows the observation of dynamic changes under minimally invasive conditions. Following the injection of Hb-Vs (at a dose of one-tenth of the circulating blood volume), the microvessels were observed to expand and to become markedly dense. The specific area of the microvessels in a bird’s-eye view increased by 20% after the injection  1]..1].In microvessels, the RBCs tend to become concentrated towards the vessel center, leaving a significant layer free of RBCs near the vessel walls. A large quantity of small-size Hb-Vs would flow through this RBC-free layer because they are small and are forced away from the vessel center  3,18]. ,18. 3,18We characterized in vitro the basic photoreactions and photosensitizer effects of Hb-Vs irradiated by a dye laser with a wavelength of 595 nm in comparison with those of human RBCs. To confirm that the photosensitizer effects of Hb-V and blood mixed with Hb-V were the same as those of blood at the same Hb concentration 10 g/dL), we mixed blood and Hb-V at a ratio of 1:1 of Hb-V to Hb. We analyzed samples in three groups: heparinized human blood dispersed in normal saline (the hBlood group); Hb-Vs dispersed in normal saline (the Hb-V group); and the 1:1 mixture of blood and Hb-V in saline (the Mixed group). Absorbance at 595 nm at various concentrations of Hb was determined using a spectrophotometer. The hBlood, Hb-V and Mixed group samples all showed the same absorbance at 595 nm at all concentrations of Hb  [1].0 g/dL, w2, with V-star, Cynosure; wavelength, 595 nm; pulse width, 0.5–40 ms). We used a securing device to maintain a fixed distance between the samples and the laser handset. The temperature of each sample before and after irradiation was measured using a high-resolution digital thermometer . After the dye laser irradiation test, fluid temperatures increased by 0.345 °C in the human blood fluid, by 0.304 °C in the Hb-V fluid, by 0.365 °C in the Mixture fluid (1:1). We used a securing device to maintain a fixed distance between the samples and the laser handset. The three sample groups showed the same change in temperature after irradiation with the dye laser, with Hb concentration maintained at 10 g/dL [To check the temperature increase with laser irradiation, we irradiated 2 mL samples from each group at an Hb concentration of 10 g/dL using constant laser parameters  at a fixed pulse width of 10 ms. The clinical endpoint goal during laser therapy was a transient gray to blue discoloration of the skin that evolved into purpura. We adopted these parameters of laser output because they were the borderline values producing skin discoloration in previous experiments [We divided the animals into three groups, irradiated at different fluences. These groups were then divided into two subgroups: control animals which did not receive Hb-V injections and intervention animals that received an intravenous injection of a bolus of Hb-V at a dose of one-tenth of the circulating blood volume. Thirty minutes after the Hb-Vs intravenous injection, we irradiated 7-mm-diameter areas of the wattle at the three fluences . This demonstrated that the severity of vascular destruction varied according to both the parameters of the laser output and whether an Hb-V injection had been administered.The distribution of these histopathological findings scores is presented in Treating PWSs remains challenging for most plastic surgeons and dermatologists because most PWSs cannot be completely cleared even after many sessions of treatment with a pulse dye laser, the treatment of choice ,7. FindiThe primary finding of our study was that Hb-Vs exhibited the same response to the dye laser as human RBCs . The Hb-Laser treatment for PWSs with Hb-V has some advantages compared with the use of other chromophores such as a self-blood transfusion. Self-blood transfusion is conducted at the bedside under stringent controls in order to maintain the freshness of the blood and to prevent a mismatch between the patient and the blood formulation . The useLaser treatment for PWSs using Hb-Vs also has some advantages compared with other chromophores, such as chemical photosensitizers. Because Hb-Vs do not leak from blood vessels into the dermis, they do not result in solar photosensitivity. In addition, the Hb-Vs and RBCs cooperate to produce thermal energy quickly. Hb-Vs can work better than chemical photosensitizers in combination with the existing light therapy equipment used worldwide, and there is no need to buy new, expensive equipment. Physicians can utilize their experience of PWS treatment that has been in practice over 30 years. These attributes of the Hb-V make it a suitable therapy method for non-malignant disorders and for pediatric patients, and its use brings economic benefits.In the treatment of PWSs with light equipment, Hb-Vs fulfill the same function as human RBCs, providing a photosensitizer effect that destroys the vessel wall. Furthermore, Hb-Vs are easier for experienced physicians to use than other chemical photosensitizers and bring benefits in medical safety and economy.


Many studies have focused on how drugs are formulated in the sol state at room temperature leading to the formation of in situ gel at eye temperature to provide a controlled drug release. Stimuli-responsive block copolymer hydrogels possess several advantages including uncomplicated drug formulation and ease of application, no organic solvent, protective environment for drugs, site-specificity, prolonged and localized drug delivery, lower systemic toxicity, and capability to deliver both hydrophobic and hydrophilic drugs. Self-assembling block copolymers  with large solubility variation between hydrophilic and hydrophobic segments are capable of making temperature-dependent micellar assembles, and with further increase in the temperature, of jellifying due to micellar aggregation. In general, molecular weight, hydrophobicity, and block arrangement have a significant effect on polymer crystallinity, micelle size, and in vitro drug release profile. The limitations of creature triblock copolymers as initial burst release can be largely avoided using micelles made of pentablock copolymers. Moreover, formulations based on pentablock copolymers can sustain drug release for a longer time. The present study aims to provide a concise overview of the initial and recent progresses in the design of hydrogel-based ocular drug delivery systems. Commonly used dosage forms, i.e. eye solutions, ointments, gels, and suspensions, have some drawbacks that might lead to poor ophthalmic bioavailability.1 Currently, there are several recommended noninvasive methods involving the use of hydrogels3 to increase ophthalmic bioavailability of drugs. Hydrogels are specific categories of polymeric networks that can soak up and retain a considerable amount of water while keeping their three-dimensional wholeness.4 Hydrogels applied for drug delivery purposes are normally made ex vivo and then saturated with drugs prior to placing the hydrogel-drug complex into the body.5Of the various routes of drug delivery, ocular drug delivery is one of the most challenging ones.6 The major shortcoming of such an approach is the necessity of the emplacement of the preformed material. Bulk hydrogels have distinct dimensions and are often highly elastic. These properties prevent their extrusion via a needle.7 The second problem may sometimes be surpassed by turning the premade gel into micro or nanoparticles.5 Hydrogels may also be formed in situ in some applications, although in these cases the possible dangers of being exposed to UV radiance or to chemicals used for cross-linking has to be checked. The later problem can be overcome using the non-cross-linked linear polymers as vehicles for drug delivery.5 Generally, the rate of drug release from these polymers is inversely related to the viscosity of the polymer matrix.8 However, it seems difficult, or even infeasible, to dissolve the polymer of choice at a sufficient amount, and thereby adjust the rate of drug release to the desired limit.5 Even if that were feasible, the high yield stress or high viscosity of the resulting substance may prevent injection or its flow through a lanky needle.9 Furthermore, extremely hydrophilic polymers swell in the aqueous environment inside the body and then dissolve, sometimes in a short time frame, unless they are partially cross-linked.10 These observations have added to the interest in formulations that display the characteristics of linear polymer solutions outside the body (letting facile injection) but convert to gel upon entering inside the body (giving a long-term drug release profile).11 The objective of this review is to give a brief introduction to stimuli-responsive hydrogels and particularly thermosensitive micelles as drug delivery vehicles. Additionally, the most recent works on ocular drug delivery using novel pentablock copolymers are discussed at the end of the review.Hydrogels can be formed using a wide variety of cross-linking techniques containing UV-photopolymerization and different chemical cross-linking procedures. Such cross-linking manners are beneficial only when the poisonous reagents are removed thoroughly before entering the hydrogel into the body. The concurrent leaching of the entrapped drug out of the hydrogel may occur during the removal of these reagents.12 In situ gel-forming formulations have the potential to be administered in liquid phase into the eye and then change into viscoelastic gel upon administration.13 Changes are made to the pH, temperature, and electrolyte compositions to make phase transition on the surface (14In situ (e.g. in the eye cul-de-sac) gel formation theory was first suggested in the early 1980s. surface .1416 An in situ gel-forming formulation has to be a low-viscose, free-flowing liquid to be easily administered into the eye as a drop, and the gel made following the phase transition needs to be strong enough to endure the shear forces existent in the cul-de-sac and display high retention time in the eye.17Since it is aqueous-based, the resulting swollen hydrogel is very convenient in the human eye.The advantages of ophthalmic in situ hydrogels would be:19reduced dose concentration and frequency, improved patient compliance, ease of application in comparison with soluble or insoluble insertions, possibility of administration of exact amount of medication, dose reproducibility, and enhanced bioavailability owing to both improved pre-corneal retention time and reduced nasolacrimal drainage of the drug.20 Polymers containing anionic (weakly acidic) and cationic (weakly basic) groups , respectively swell and shrink in response to increases in the external pH.21pH-sensitive polymers include pendant alkaline or acidic groups that receive or release protons due to the changes in the pH of medium. The polymers with lots of ionizable groups are called polyelectrolytes.1 The instilled solution changes into gel due to a change in the ionic strength. The rate of electrolyte absorption by the polymer from the tear fluid depends on the osmotic gradient across the gel surface. Therefore, the rate of sol transition into the gel is probably influenced by the osmolality of the solution. The electrolytes naturally found in the tear fluid, particularly Ca, Na, and Mg cations, induce polymers to form a gel when it is applied as a flowing solution into the cul-de-sac.2Ion-stimuli polymers concern the generally applied in situ gelling materials for ophthalmic drug delivery.22 For simplicity, temperature-sensitive hydrogels have been categorized into three classes—positively thermosensitive, negatively thermosensitive, and thermally reversible gels.23Temperature-sensitive hydrogels are a group of polymeric systems that are sensitive to environmental factors. These hydrogels can swell or shrink in response to any changes in the surrounding liquid temperature.25 Copolymers of N-isopropylacrylamide (NIAAm) display an ‘on/off’ drug release26 with the ‘on’ state at a lower and the ‘off’ state at a higher temperature than LCST, and give a pulsatile scheme to drug release.27 Generally, LCST systems are utilized to control the release of drugs, particularly proteins.29 Liposomes that thermosensitive polymers have stabilized on their membrane can release their content in a controlled manner.30 Bulmus et al. utilized PNIPAAm polymers, conjugated to a particular site near the biotin-binding site of streptavidin, for ‘on/off’ control of biotin access to its binding site.31 Below the LCST, i.e. 32°C for PNIPAAm, the polymer is in its completely extended conformation due to desired interaction with water molecules. In this conformation, the biotin-binding site on streptavidin is accessible to interact with biotin. However, above this temperature, the polymer collapses, preventing biotin accessibility to its binding site.32Negatively thermosensitive hydrogels, having a lower critical solution temperature (LCST), collapse or shrink upon an increase in temperature above the LCST and swell upon a decrease in temperature below the LCST.33 Polymer lattice of polyacrylamide (PAAm)1, poly (acrylamide-co-butyl methacrylate),34 and poly (acrylic acid) (PAA)35 possess positive thermosensitivity of swelling. The transition temperature of P(AAm-co-BMA) shifts to a higher temperature with increasing butyl-methacrylate content of copolymer.32 Aoki et al. fabricated an UCST system using Poly combined with Poly(acrylic acid).36Positively thermosensitive hydrogels, having an upper critical solution temperature (UCST), collapse or shrink upon a decrease in the temperature below the UCST and swell upon an increase in the temperature above the UCST.37 These polymers make a free-flowing solution at room temperature that can be converted to gel at body temperature.38 Such a system can be conveniently injected into the body cavities.39 In some cases when decreasing the amount of the thermogelling polymer is cost effective or necessary, it can be possible to decrease the total amount of thermogelling polymer by mixing with a reversible gel-induced polymer that is sensitive to pH.28 New classes of biodegradable triblock copolymers have been developed. The polymers containing poly (ethylene glycol)-poly-(D-L lactic acid-co-glycolic acid)-poly(ethylen glycol) (PEG-PLGA- PEG)40 or PLGA-PEG-PLGA42 were studied as injectable sustained drug delivery systems. Certain natural polymers such as xyloglucan can also be used in the formation of thermoreversible gels.43Most of the currently applied thermoreversible gels are produced by poly (ethylene oxide)-b-poly (provpylene oxide)-b-poly (ethylene oxide) .44To explain the sol-gel phase transition after an increase in the temperature, three main mechanisms have been suggested—gradually losing the water of hydration  micelles.53 The hydrophilic parts of block copolymers form the external shell of polymeric micelles and play a significant role in their in vivo behaviour, particularly for their steric consolidation and the capability to interplay with cells.54 The conformation of polymer in solution is affected by the lengths of the hydrophilic and hydrophobic segments, so that longer hydrophilic blocks cause polymers to keep in a monomeric state in water.55Typically, hydrophobic parts of the block copolymers form internal core of the polymeric micelles via hydrophobic interaction56 Dynamic light scattering (DLS) is the widely used method for determination of the hydrodynamic diameter of polymeric micelles.58 Different types of methods like conductivity, interfacial tension, and osmotic pressure are utilized for the assessment of CMC.59 However, since the CMC values of polymeric micelles are very low, these techniques may not be useful in these cases. Light scattering is a powerful technique; however, it can be applied to portend the outset of micellization, only if the CMC happens in a range of concentrations that this method is sensitive to.60 Adsorption of polymer in the column is one of the problems that restrict the use of gel permeation chromatography (GPC) in determining CMC.61 One of the best choices for the assessment of CMC in polymeric micelles is pyrene fluorescence. The fluorescence spectrum of pyrene display particular bands near 370–400 nm, whose relative and absolute intensities, positions, and widths are highly dependent on the polarity of its microenvironment.63 Following the increase in polymer concentration, the intensity ratio of the first and third bands (I/III ratio) decreases tremendously due to changes in the polarity of pyrene environment.65 This reduction occurs owing to the accumulation of pyrene as a hydrophobic probe in the apolar micellar core around the CMC.66 Hence, we can easily determine the CMC by plotting the II/IIII ratio against polymer concentration. The junction of the slope tangent and the lower horizontal is known as the CMC of the system.67Micelles are determined by measuring the turbidity, particle size, and CMC. Ionic micellar dispersals become turbid at a higher temperature than nonionics do. The clouding aspect is an undeviating consequence of the formation of larger particles.68  (PLGA),83 poly(caprolactone) (PCL),84 and poly(lactide) (PLA)85 have been extensively considered for the provision of nanoparticles. In particular, amphiphilic copolymers with polyethylene glycol as their hydrophilic segment such as PCL–PEG,86 PLGA–PEG,87 PLA–PEG,88 and PCL–PEG–PCL89 have been considered in controlled drug delivery. PEG is well known due to its nontoxicity and absence of antigenicity.90 Furthermore, PEG mediates the drug release via a diffusion mechanism by facilitating the penetration of water into nanoparticles. PCL is an FDA-approved, biodegradable, and nontoxic polyester that is miscible with a variety of polymers and has high permeability to small drug molecules.92 In addition, due to its hydrophobic nature, it is very capable of encapsulating lipophilic drugs through hydrophobic interactions. However, its application is limited because of its high crystallinity and hydrophobic nature, which results in a very slow degradation rate.93 PCL- and PEG-based triblock copolymers such as PEG–PCL–PEG or PCL–PEG–PCL have been extensively studied for drug delivery. The releasing profile of the drug is greatly sustained by increasing the molecular weight of PCL block. However, high molecular weight PCL block enhances the total hydrophobicity and crystallinity of the polymer, thereby causing the initial burst release of the nanoparticles made from such triblock copolymers.94 Hence, there is still a need for optimized block copolymers that can sustain drug release over a longer time without significant initial burst release.In recent years, many researchers have investigated the use of nanoparticles in ocular drug delivery.et al. studied the injectable and biodegradable thermosensitive in situ gels for sustaining delivery of protein drugs in the treatment of ophthalmic posterior disease. They synthetized a series of triblock (TB) and pentablock copolymers (PBCs) of PCL-PEG-PCL, PLA-PCL-PEG-PCL-PLA and PEG-PCL-PLA-PCL-PEG, and investigated the effects of hydrophobicity, block arrangement, and molecular weight on the crystallinity of copolymer. Results of sol gel transition studies confirmed that aqueous solutions of block copolymers can convert to gel upon exposure to body temperature. Although both tri and pentablock copolymers could prolong the release of IgG, it was significantly longer for pentablock copolymers. Furthermore, the syringeability of PEG-PCL-PLA-PCL-PEG pentablock copolymer was better than both PCL-PEG-PCL and PLA-PCL-PEG-PCL-PLA copolymers due to the lower kinematic viscosity of its aqueous solution at 25°C. The crystallinity of both PBCs were lower than TBC because of the presence of PLA blocks, and therefore, it was expected that the rate of degradation of PBCs would be faster than that of TB copolymer.95Patel 96They also synthetized and evaluated a PB copolymer comprising PEG, polyglycolic acid (PGA), PCL, and PLA for controlled delivery of FITC-BSA, IgG, and bevacizumab in the treatment of posterior eye diseases. They studied the effect of different ratios and various molecular weights of blocks on the release profile. They showed that both the hydrophobicity of the copolymer and the hydrodynamic diameter of the loaded protein have a momentous effect on EE (entrapment efficiency) and release profile. Their studies also demonstrated that, while the nanoparticles display sustained release profile with an initial burst release, it is possible to reach a near zero order pattern of release with no or slight burst release by suspending NPs in a thermosensitive gel.97In another study, they designed a series of PBCs based on PGA-PCL-PEG-PCL-PGA and PLA-PCL-PEG-PCL-PLA for sustaining delivery of IgG as a model protein. They studied the effect of polymer composition, molecular weight and isomerism on drug loading (DL), entrapment efficiency (EE), and in vitro release profile. Molecular weight and the crystallinity of copolymers indicated a considerable effect on these parameters. They moderated the crystallinity of PBCs by altering the ratios of PLA/PCL or PGA/PCL blocks, besides using different isomers of PLA . PBCs consisted of PLA, with D,L-lactide displaying higher EE and slower release profile compared to PB copolymers comprising PLA with L-lactide or PGA.in vitro release rate was inversely proportional to these parameters.98They also synthetized a series of PBCs using PCL, PEG, and PLA or PGA, and entrapped various proteins/peptides into the prepared copolymers though the double emulsion solvent evaporation method. In order to reach a constant zero order release profile and decrease the burst release to the lowest amount, they used a novel composite conception by suspending the protein/peptide-loaded PB nanoparticles in thermosensitive PB gel. The authors investigated the influences of various parameters on DL, EE, and in vitro release profile. The results showed that an increase in molecular weight of copolymer, as well as a decrease in the volume of external phase, would enhance both DL and EE. However, the addition of salt either in the external or internal phase had a small effect on EE. Besides, while there was a direct proportion between molecular weight/hydrodynamic diameter of biotherapeutics and the resulted DL or EE, the et al. synthetized a PBC comprising PLA-PCL-PEG-PCL-PLA for sustaining the release of steroids over a longer time interval. They investigated the effect of incorporation of poly (L-lactide) (PLLA) or poly  (PDLLA) on the crystallinity of PBCs and the in vitro release profile of triamcinolone acetonide as a model drug from nanoparticles. The results showed that the incorporation of suitable ratio of PDLLA in the existent PCL-PEG-PCL copolymers lowered the crystallinity of copolymer and considerably minimized the initial burst release from NPs. The authors suggested that nanoparticles made from PBCs can minimize the limitations of TBC nanoparticles such as initial burst release and can sustain the release of drug for a longer time.93Tamboli et al. designed a pentablock copolymer, PLA–PCL–PEG–PCL–PLA, to develop pazopanib-loaded nanoparticles for use in the treatment of ocular neovascularization. They studied the effect of incorporation of pazopanib (a substrate of efflux transporters) in nanoparticles on bypassing the drug efflux system .The prepared nanoparticles prolonged the delivery of pazopanib by up to 100 days without any remarkable burst release and succeeded in evading the efflux transporters99Khurana et al. have published their research on developing a PB copolymer composite comprising PCL-PLA-PEG-PLA-PCL IgG-Fab-loaded NPs suspended in thermosensitive mPEG-PCL-PLA-PCL-PEGm gel. Using this composite formulation, they could sustain the release of macromolecules over 80 days with negligible initial burst release occurrence. The size of the prepared NPs was 150 nm and % EE and % Dl were 66.64% ± 1.75 and 18.17% ± 0.39, respectively. The biocompatibility studies implemented on ocular  and macrophage (RAW 264.7) cell lines indicated the safety of the PB copolymer-based composite formulations for clinical uses.100Recently, Agrahari In situ gel-forming systems are potential ocular delivery systems as they can overcome the shortcomings associated with common ocular dosage forms. Therefore, they have received much attention in recent years. Drug-incorporated liposomes, nanoparticles, micelles, etc., can also be suspended in these systems to achieve highly effective and sustained drug delivery. The limitations of available triblock polymers such as initial burst release can be largely avoided by using micelles made of pentablock copolymers. In addition, formulations based on pentablock copolymers can sustain drug release for a longer time. Thus, novel pentablock copolymers are good materials that may be used as a carrier for ophthalmic drug delivery as well as for other illnesses that need sustained drug delivery.The financial support from Drug Applied Research Center and Research Council of Tabriz University of Medical Sciences is greatly acknowledged.Not applicable.The authors have no conflicts of interest to declare.


Background/Objectives. Balance problems are well-established modifiable risk factors for falls, which are common in older adults. The objective of this study was to establish the efficacy of a Wii-Fit interactive video-game-led physical exercise program to improve balance in older Veterans. Methods. A prospective randomized controlled parallel-group trial was conducted at Veterans Affairs Medical Center. Thirty community dwelling Veterans aged 68 (±6.7) years were randomized to either the exercise or control groups. The exercise group performed Wii-Fit program while the control group performed a computer-based cognitive program for 45 minutes, three days per week for 8-weeks. The primary ) and secondary outcomes  were measured at baseline, 4 weeks, and 8 weeks. Results. Of 30 randomized subjects, 27 completed all aspects of the study protocol. There were no study-related adverse events. Intent-to-treat analysis showed a significantly greater improvement in BBS in the exercise group  compared to the control group  at 8 weeks (average intergroup difference (95% CI), 5.5 (4.3–6.7), p < 0.001) after adjusting for baseline. Conclusion. This study establishes that the Wii-Fit exercise program is efficacious in improving balance in community dwelling older Veterans. This trial is registered with ClinicalTrials.gov Identifier NCT02190045. Gait and balance problems increase with age, making older adults vulnerable to falls. Community dwelling adults, aged 65 years or older, have a 22% yearly incidence of falls . Falls aExercise improves gait and balance in the elderly. A Cochrane review found statistically significant improvement in balance following exercise interventions compared to usual activity . AnotherExergames are interactive video games involving physical activity or exertion tracked as body movement . The tecThis pilot study was an 8-week, prospective randomized controlled parallel-group trial comparing the effects on measures of balance in the exercise group (Wii-Fit program) to that in the control group (Brain-Fitness program). The study was conducted at Department of Veterans Affairs Medical Center. The protocol was approved by the Institutional Review Board of the Central Arkansas Veterans Healthcare System. Thirty subjects were recruited to inform sample size calculations for a larger study. After responding to advertisements, recruits were prescreened by medical records review to determine eligibility. Community dwelling adults aged ≥ 60 years were included in the study. Subjects using wheel chairs or walkers for mobility, having absolute contraindications to exercise per American College of Sports Medicine guidelines, and those with medical conditions that in the opinion of the study physician were likely to compromise safe study participation were excluded .n = 15) and control groups (n = 15) using sealed envelopes prepared by the statistician. Research assistants monitored participation in both groups for exercise fidelity, safety, and compliance. Participation time and activities performed were recorded. Subjects were allowed to choose the activities performed for Wii-Fit or Brain-Fitness though research assistants encouraged progression to higher level and inclusion of activities from each category, described below, at each session.Subjects who cleared the prescreening were invited for the baseline visit during which they provided a written informed consent. At this visit, eligibility was further assessed with Berg Balance Scale (BBS) and Mini Mental State Exam (MMSE). Those that scored BBS ≤ 52 and MMSE ≥ 24 were included in the study. The cut-off score of 52 on BBS was selected in an effort to avoid ceiling effects and to include those with mild-to-moderate balance problems. Demographics and anthropometric data were collected at the baseline visit. Eligible subjects were trained to use Wii-Fit and Brain-Fitness programs over one to two sessions to ensure they were willing and capable of safe participation in either program. Subjects were randomized using a randomized block design to the exercise , lateral weight shifting , multidirectional balance , and multidirectional balance with a cognitive component (Perfect 10). Subjects tracked their activity and progress using an individualized icon . Each session included a warm-up, exercise, and cool-down phase. During the warm-up and cool-down phases, subjects walked for five minutes at a self-selected comfortable pace using the program's “basic walk” activity. The exercise phase was designed to be patient centric. Instead of doing exactly the same exercises, subjects were encouraged to choose one or more exercises from every Wii-Fit category during each session. Research assistants were in the exercise room during the entire session maintaining visual contact and being readily available if necessary.Subjects performed cognitive exercises using a commercially available computer program, Brain-Fitness  , 17 for All outcomes were measured at baseline and four and eight weeks after intervention initiation. The primary efficacy outcome was the Berg Balance Scale (BBS), which assesses balance impairments in older adults and is a good measure of static and dynamic stability . It consThe secondary outcomes included Activities-Specific Balance Confidence (ABC) scale, Physical Activity Enjoyment Scale (PACES), Modified Mini Mental State Exam (3MS), Rand Short Form 36 (SF-36), and incidence of adverse events. The ABC scale measures fear of falling and self-confidence to maintain balance . Subject3MS is a global screen for cognitive function that covers orientation to time and place, registration, recall, simple language, and construction. Test-retest reliability ranges from 0.91 to 0.93 . The tot t-tests or nonparametric Wilcoxon rank sum tests (continuous data) and Fisher's exact tests . Changes from baseline at 4 and 8 weeks in primary and secondary outcomes were analyzed using repeated measures mixed model analyses of covariance. Group (exercise and control) and time (4 and 8 weeks) as well as the interaction between the two were independent variables; the dependent variable's baseline measure was included as a covariate. In post hoc analyses, groups were compared in terms of the changes from baseline at 4 and 8 weeks using t-tests derived from model-based contrasts. Additionally, comparisons were made to separately assess improvements within each group. A sensitivity analysis to investigate bias due to missing data for subjects (n = 3) who dropped out of the study was performed by carrying forward baseline values to conservatively impute missing data as no change; we also investigated the impact of including MMSE and SF-36 Physical and Mental summary scores as covariates in the sensitivity analysis. Two-sided p values less than 0.05 indicated statistical significance. Data were analyzed using SAS Enterprise Guide v5.1 .Subject demographics and baseline characteristics were compared usingBalance Games were the most accessed exercises in the exercise group (Ski Jump (median 11 of 24 visits), followed by Bubble Balance, Penguin Slide, and Table Tilt ). Simple Yoga poses , Strength Training , Training Plus , and Balance Games  were chosen at least once by at least 50% of the participants. Some of the outcomes could depend on the game preferences, the details of which are included in N = 27), there was a significant group-by-time interaction for the primary outcome measure BBS  at 4 weeks and 5.5  at 8 weeks. Within-group analysis showed significant improvement in BBS in the exercise group at 4 weeks  and at 8 weeks  but no improvement in the control group at 4 weeks  or at 8 weeks . All subjects in the exercise group who completed the study demonstrated a clinically relevant change of greater than or equal to four on the BBS at 8 weeks [p = 0.001) at 4 weeks and 4.1  at 8 weeks. Adjusting for MMSE and SF-36 Physical and Mental summary scores resulted in between-group differences that were only slightly (<10%) larger, and these covariates were nonsignificant .For the subjects who completed the study ( 8 weeks . Additiop = 0.046)  but not at 8 weeks. However, the group-by-time effect was nonsignificant for these variables. There were no significant group-by-time interactions or between-group differences for any of the other secondary outcomes.There was a significant average improvement (95% CI) within the exercise group at 8 weeks on the secondary outcome measure, ABC scale  . Analysip = 0.08). Additionally, our analyses adjusted for baseline scores to further validate the effects of the program on balance. The study was not a priori powered to detect differences in secondary outcomes.The study sought to establish the efficacy of Wii-Fit exercise for improving balance in older adults with mild-to-moderate balance problems. As in prior studies, participants in this study experienced no adverse events, indicating that older adults with comorbidities can safely complete the program. The study also verified that Wii-Fit is feasible to use for training since the adherence rate was high and participants were able to exercise independently. Efficacy was demonstrated for the primary outcome, BBS. The average BBS change was six points and all subjects improved by at least four points at 8 weeks, a clinically relevant change observed in a similar community dwelling population . Change Balance-board-based exercises have been used to improve balance . Wii-FitAs described in recent reviews, 35 prior studies examined the use of Wii-Fit or other balance-board-based exercise games to improve balance or motor function , 36. Of Wii-Fit exercises are shown to improve motor function independent of balance. Two RCTs of Wii-Fit demonstrated statistically significant between-group differences on motor function in select populations of older adults. Saposnik et al. demonstrated significant improvement in the motor function in poststroke subjects in the Wii-Fit augmented standardized rehabilitation therapy group compared to the recreational therapy group . JorgensThe current study adds to the findings of the prior studies by demonstrating that Wii-Fit produced a significantly greater effect on balance compared to the control intervention after controlling for baseline BBS scores. This is perhaps due to the differences in the dose of the intervention or other aspects of study design compared to previous studies. This includes analyzing the primary outcome measure of balance using intent-to-treat principles in a sensitivity analysis, adjusting for baseline, and having an attention control group. There were several other strengths of the study. First, 30% of the subjects were African Americans, which make the results more generalizable. Second, there was a high adherence to the program. In addition, none of the subjects stopped exercising before the time allotted for them to exercise; this suggests that making the program self-guided and tailored to comply with each subject's preferences and abilities was effective in keeping the subjects engaged. Third, we clearly demonstrated that subjects were able to safely participate in the Wii-Fit program with no study-related adverse events.The current study has several limitations. Most notable one was the limited sample size. The study was a pilot investigation and was not a priori adequately powered to address all of the outcomes. The study population primarily consisted of male Veterans; although consistent with the older population enrolled within the Veterans Health Administration, this limits the generalizability. Another major limitation was the lack of blinding of the outcomes assessor. The assessor was not part of the exercise sessions; however, the assessor was aware of the group assignment. The use of Wii-Fit as an exercise modality was a limiting factor for morbidly obese subjects as the Wii balance board has an upper weight limit. Activity levels between exercise sessions were not recorded. Although there is no reason to believe that participants in either group were more active than others in between the sessions, such information could help explain the outcomes. Another limitation of the study design was the lack of postintervention follow-up to study the retention of benefits of exercise. The duration of the exercise program in our study was 8 weeks, shorter than the recommended duration of 12 weeks, which is reported to have the largest effect on balance . The addThis study confirms the safety and feasibility of a Wii-Fit exercise program for community dwelling older adults with mild-to-moderate balance problems. Furthermore, it also demonstrates that Wii-Fit exergames can be used efficaciously in this population to improve balance.


Although competing endogenous RNAs (ceRNAs) have been implicated in many solid tumors, their roles in breast cancer subtypes are not well understood. We therefore generated a ceRNA network for each subtype based on the significance of both, positive co-expression and the shared miRNAs, in the corresponding subtype miRNA dys-regulatory network, which was constructed based on negative regulations between differentially expressed miRNAs and targets. All four subtype ceRNA networks exhibited scale-free architecture and showed that the common ceRNAs were at the core of the networks. Furthermore, the common ceRNA hubs had greater connectivity than the subtype-specific hubs. Functional analysis of the common subtype ceRNA hubs highlighted factors involved in proliferation, MAPK signaling pathways and tube morphogenesis. Subtype-specific ceRNA hubs highlighted unique subtype-specific pathways, like the estrogen response and inflammatory pathways in the luminal subtypes or the factors involved in the coagulation process that participates in the basal-like subtype. Ultimately, we identified 29 critical subtype-specific ceRNA hubs that characterized the different breast cancer subtypes. Our study thus provides new insight into the common and specific subtype ceRNA interactions that define the different categories of breast cancer and enhances our understanding of the pathology underlying the different breast cancer subtypes, which can have prognostic and therapeutic implications in the future. Non-coding RNAs are a class of RNAs that regulate gene expression transcriptionally and post-transcriptionally . The miRBreast cancer is a progressive disease that is a leading cause of cancer-related mortality in women and composed of distinct subtypes that are highly heterogeneous –10. BaseAlthough many breast cancer biomarkers have been reported , 19, it Therefore, the aim of this study was to generate subtype specific dysregulated miRNA mediated ceRNA networks to discover new critical subtype related genes and to unravel the shared and specific pathogenic pathways of the breast cancer subtypes at a system levelTo systematically identify disease markers in each of the four breast cancer subtypes, we performed Wilcoxon Rank Sum tests to identify differentially expressed protein coding and non-coding genes in each of the breast cancer subtypes Figure . Each suRegarding miRNAs, previous studies have suggested that miRNAs usually function as negative regulators of gene expression , 26. TheTo investigate the ceRNA regulation in breast cancer subtypes, we generated a ceRNA network for each subtype by assembling all the significant ceRNA interactions that were based on the significance of both, their positive co-expression and the shared miRNAs, in the corresponding subtype miRNA dys-regulatory network Table . AccordiSeveral marker genes of specific breast cancer subtypes have been reported to be differentially expressed , 11. We A key network property is node connectivity that shows the degree of communication among the nodes. Hub node is pivotal and shows high connectivity with other nodes. In our study, we defined the hubs as the top 10% of the nodes with the highest degree within the networks. The hub nodes were sub-divided into subtype common and subtype specific ceRNA nodes that would identify mechanisms that are either shared or specific among the different breast cancer subtypes. The subtype common ceRNA hubs were defined as ceRNA hubs that occurred in at least three of the subtype ceRNA networks and accounted for 12.65%, 29.07%, 39.34% and 19.39% of total ceRNA hubs in luminal A, luminal B, HER2-enriched and basal-like, respectively Figure . On the While the node degree of a network represents the local centrality, k-core decomposition reveals the modularity of central hub nodes and is used to identify the largest sub-graph where the vertices have at least ‘k’ connections within a subgraph called as the k-cores . TherefoNext, we analyzed the relationship among common ceRNA hubs to understand their regulation of breast cancer progression. Towards this, we collected all the ceRNA interactions among the 28 common ceRNA hubs in the four breast cancer subtypes to form a subtype common sub-network Figure . Five ofFurthermore, our data showed that the lncRNAs functioned as ceRNAs involved in the cancer related biological processes. We found RP11-276H19.1 (ENSG00000226237) as a common hub that participated in the ceRNA interaction networks in luminal A, luminal B and HER2-enriched subtypes. It also co-regulated with MSRB3 and LHFP by sharing miRNAs to form ceRNA-ceRNA interactions in the three subtypes Figure . Also, tTo discover the pathogenic mechanisms of different breast cancer subtypes, we further focused on the subtype specific ceRNA hubs. We extracted four subtype-specific ceRNA hub sub-networks by collecting all subtype-specific ceRNA hubs and their ceRNA interactions within each of the four subtype ceRNA networks and found that the subtype-specific hubs were highly connected with each other and regulated by hub miRNAs in the miRNA dys-regulatory network of each subtype Figure . SeveralBased on these data, we hypothesized that the subtype-specific ceRNA hubs that took part in subtype specific pathways could be used to classify different breast cancer subtypes. We also performed the principal component analysis (ggfortify R package) and found that the 29 subtype-specific ceRNA hubs that were dysregulated among all breast cancer subtypes  could distinguish the basal-like, HER2-enriched and luminal subtypes, whereas distinguishing the luminal A and B subtypes was not clear-cut Figure .Taken together, these results suggested that investigating the properties of ceRNA interaction networks may help strengthen our understanding of different subtype breast cancers, which may contribute to breast cancer subtype phenotype, thus further to improve clinical prevention and treatment.In this study, we developed a computational framework to construct and analyze ceRNA networks using the transcriptional profiling data to understand the dynamic clinical behaviors and outcomes of different breast cancer subtypes.-12, 1.18e-04, 7.11e-09, respectively, suggesting that the ceRNA interactions were reliable. We further validated the accuracy of predicting the ceRNA interactions globally by analyzing expression datasets for the four breast cancer subtypes from another array, GSE45827, and found that the ceRNA interaction pairs showed higher positive co-expression compared with the gene pairs that were randomly selected  for the luminal A, luminal B, HER2-enriched, basal-like and normal-like, with normal breast samples [The mRNA and miRNA expression datasets measured by RNA sequencing were downloaded from The Cancer Genome Atlas (TCGA) , whereas samples . As the  samples , it was To obtain the regulatory relationships between miRNAs and mRNAs (or lncRNA), miRanda algorithm was used to predict the miRNA-target relationships. As a result, 1,129,584 unique miRNA-lncRNA and 2,927,669 unique miRNA-mRNA relationships were generated among 1,017 miRNAs, 7,731 lncRNAs and 17,215 mRNAs, respectively.Functional annotation gene sets were downloaded from the Molecular Signatures Database (MsigDB) . This inA three-step model was proposed to construct ceRNA networks. Firstly, we identified differentially expressed mRNA, miRNAs and lncRNAs for each of the breast cancer subtypes compared with normal breast samples. Secondly, four miRNA dys-regulatory networks were constructed by considering negative regulation between dysregulated miRNAs and their targets. Thirdly, based on the miRNA dys-regulatory networks, ceRNA interactions were identified by considering significant sharing of the the regulatory miRNAs and their co-expression. All ceRNA interactions were assembled into a ceRNA network for each breast cancer subtype, where the nodes were referred to as ceRNAs and the edges referred to as ceRNA interactions.Wilcoxon Rank Sum tests were used to identify differentially expressed protein coding genes and non-coding genes between each subtype and normal breast samples. We applied the Bonferroni correction and the RNAs that showed a FDR less than 0.05 and the absolute log2 fold change (LFC) no less than 1 were considered significant.Differentially expressed target genes (mRNAs and lncRNAs) for each differentially expressed miRNA were collected and a miRNA dys-regulatory network was constructed for each subtype by considering negative regulation between miRNAs and their targets based on the expression profiles of each breast cancer subtype samples .The subtype ceRNA interaction networks were constructed based on the miRNA dys-regulatory networks. Two target genes were considered to be competing only if 1) they significantly shared regulatory miRNAs in the miRNA dys-regulatory network as calculated by the hypergeometric test  and 2) the expression of ceRNA-ceRNA interactions showed a statistically significant positive correlation with a Pearson correlation coefficient that was no less than 0.5 . All the ceRNA interactions were assembled into a ceRNA network for each breast cancer subtype and the weights of all edges were set to 1.The k-core is the largest sub-graph where every node has at least k links. The k-core can be determined by pruning all the vertices from a network with degrees less than k and their incident links until the degree of all the remaining vertices in the sub-graph are larger than or equal to k. The k layers reflect the connectivity of the ceRNA partners such that the first peeled layer (k=1) contains ceRNAs with only a few connections whereas the last peeled layer is most connected. Each node was assigned to a maximal k core sub-network in each breast cancer subtype ceRNA network. For among different breast cancer subtypes, the layers of each ceRNA network were normalized to 0-1 divided by the number of largest k layer in each network.To dissect the biological pathways represented by the common or subtype-specific ceRNA hub sub-networks, functional enrichment analysis of the hub ceRNA genes was performed by hypergeometric distribution with the Bonferroni correction (FDR < 0.05).


Ptrend = 0.002; Me-Can, Ptrend = 0.04) and pre-diagnostic diabetes . During the year before diagnosis, blood glucose was inversely associated with glioma in the AMORIS  but not the Me-Can cohort . This AMORIS result is consistent with our hypothesis that excess glucose consumption by the preclinical tumour accounts for the inverse association between blood glucose and glioma. We discuss additional hypothetical mechanisms that may explain our paradoxical findings.Previous literature indicates that pre-diagnostic diabetes and blood glucose levels are inversely related to glioma risk. To replicate these findings and determine whether they could be attributed to excess glucose consumption by the preclinical tumour, we used data from the Apolipoprotein MOrtality RISk (AMORIS)  and the Metabolic syndrome and Cancer project (Me-Can) cohorts . We identified individuals who were followed for a maximum of 15 years after their first blood glucose test until glioma diagnosis, death, emigration or the end of follow-up. Hazard ratios (HRs), 95% confidence intervals (CIs) and their interactions with time were estimated using Cox time-dependent regression. As expected, pre-diagnostic blood glucose levels were inversely related to glioma risk (AMORIS,  Symptoms usually occur only three months before diagnosis2; it is therefore important that indicators of the preclinical tumour be identified before the onset of symptoms.Glioma is a heterogeneous primary brain tumour for which there is no treatment that ensures long-term survival. Patients diagnosed with the most common adult form of this tumour, glioblastoma, survive on average only 14 months3, 11 out of 16 previous studies  would be expected to have higher all-cause mortality rates than would people with normal blood glucose levels. Thus people with hyperglycaemia are selected out of the population at risk of glioma making etiological inferences based on comparisons of the prevalence of hyperglycaemia between people with and without glioma invalid. To determine whether this potential source of selection bias is present, we estimated the modifying effects of age at diagnosis on the association between blood glucose levels and glioma. Competing risk bias would be expected to increase with age as do all-cause mortality rates. To further evaluate our data for the presence of competing risks, we used the sub-distribution hazards model23 which is a more formal method of identifying competing risks.Our rationale for evaluating age at diagnosis as a modifying factor is based on results of a previous AMORIS study of pre-diagnostic serum glucose levels and prostate cancer riskIn Table P-values in Table P-values primarily reflect the hazard ratios of the highest level of glucose (≥6.1 mmol/L) . Results restricted to high grade glioma , although it is based on only 12 glioma cases , 11 of which were found among men. There were 39,404 people  diagnosed with diabetes among the non-cases, men accounted for 25,392 of these diagnoses. Information on pre-diagnostic diabetes diagnoses was not available for the Me-Can cohort.Ptrend = 0.04) over the levels of time before diagnosis. Thus the time of blood draw modifies the effect of glucose on the glioma rate, that is, there is a statistically significant interaction between time and glucose. There is no evidence of a similar effect of time before diagnosis in the Me-Can cohort. In addition, in an exploratory analysis of within-person time trends in 51 Me-Can patients with multiple blood glucose measurements, we found no trends by year before glioma diagnosis.Table P < 0.05) modifies the association between glucose and glioma risk among men older than age 70 years in the Me-Can cohort . In addition, the initial significance tests reflected the statistical significance of a single interaction term (stratified on the second time-related term). These tests are not as meaningful as the interaction analyses presented in Tables 23. Given this limitation, the strong inverse age-blood glucose association among people older than age 70 years in the Me-Can cohort may indicate the presence of competing risks from hyperglycaemia-associated deaths. However if our findings are attributable to competing risks one would expect the inverse diabetes-cancer relationship to be observed at cancer sites other than glioma. In fact, we see the opposite pattern. Tumours at most sites are positively associated with blood glucose level26 with the known exceptions of meningioma in one study27 but not in another9 and prostate cancer28.The sub-distribution hazards model indicates the absence of competing risks; however, the weakness of this analytic approach is that although people who die are retained in risk sets after their deaths, the number of people diagnosed with glioma remains the same29 and glioma10 and therefore qualify as potentially confounding variables. Also, there are several differences between the AMORIS and Me-Can cohorts that may affect comparisons. Serum glucose was measured in the AMORIS cohort while plasma glucose was measured in the Me-Can cohort. However Frank et al.30 found no physiologically relevant differences between these two methods of measuring circulating glucose. Another difference between these cohorts is that non-fasting serum glucose values were included in the AMORIS cohort but were not included in the Me-Can cohort although fasting and non-fasting glucose values in the AMORIS cohort were similar. In addition, Me-Can study participants were censored after diagnosis of any malignant tumour while AMORIS cohort participants were not. Despite these differences, cohort dose-response findings for the two cohorts are remarkably similar. Whether conflicting estimates of time and age interactions with blood glucose levels produced by the two cohorts are attributable to their differences in design is not known.Limitations of our study include the lack of sufficient information on body mass index, blood pressure and high and low density lipoprotein in the AMORIS cohort to include these variables in our regression models. These variables are associated with both diabetes31 and may therefore reduce glioma risk. However, a large case-control study found no clear evidence of this association7.There are several potential mechanisms accounting for the inverse association between hyperglycaemia and glioma. The commonly prescribed anti-diabetic medication, metformin, inhibits glioblastoma cell proliferation and migration32. Another possibility is the fact that both impaired glucose tolerance33 and diabetes36 lead to the shortening of telomeres . The highest glioma risk alleles near the TERC and TERT gene are associated with long telomeres37 thus providing a potential rationale for our findings. The problem with this line of reasoning, however, is that one would expect hyperglycaemia to be inversely related to malignant melanoma, the risk of which is also positively associated with telomere length38, but it is not39.Adverse effects of hyperglycaemia and diabetes that may paradoxically reduce glioma risk include diabetes-related reduction of bioavailable testosterone levels40. To the extent that glioblastoma stem cells are related to neural stem cells41 hyperglycaemia may confer protection against glioblastoma. Another potential mechanism is inhibition of cerebral circulation associated with diabetes43 causing reduced glioma risk44. In addition, lower levels of insulin-like growth factor 1 (IGF-1) are found in diabetic than in non-diabetic serum45 and tumour-associated macrophage derived IGF-1 may drive glioma recurrence46. These proposed mechanisms may work together or separately.Hyperglycaemia induces apoptosis and inhibits progression of neural stem cells11 was mixed with the AMORIS data suggesting an inverse association during the year before glioma diagnosis and the Me-Can data showing no such association. Further investigation of a pre-diagnostic Warburg effect would be worthwhile, as would examination of associations between blood glucose level and glioma by genetic subtypes of type 2 diabetes47 and glioma48.Overall, our findings provide further evidence of an inverse association between hyperglycaemia, diabetes and glioma. In contrast, evidence for the presence of the Warburg effect51. Here we briefly summarise this information.The AMORIS and Me-Can cohorts have been described in detail elsewherest, 2011.This Swedish cohort links records from the Central Automation Laboratory  database (1985–1996) to those of the National Cancer Registry, the National Patient Register, the Cause of Death Register, and the National Register of Emigration. Endpoint ascertainment is based on the Swedish National Cancer Registry and therefore includes all glioma diagnoses among Swedish residents. Outcome data used in the present analyses are current as of December 3152. We then excluded 528 people diagnosed with meningioma (defined by pathology-anatomy diagnosis (PAD) codes  461 and 466) and 15 with ependymoma . These additional exclusions reduced the sample to 528,580 participants, 604 of whom were subsequently diagnosed with glioma (PAD codes 475 (n = 128) and 476 (n = 476) and the International Classification of Disease 7th Edition (ICD-7) code 193)  or death, emigration, or the end of follow-up (outcomes leading to censoring) were excluded from the study as their tumours may differ from those found in adults53 established in 1996 and the National Patient Register which began including outpatient diagnoses in 2001. The AMORIS study began in  1985 and the data set used in the present study ended in 2011 so information on diabetes was not complete, but presumably equally incomplete for glioma cases and non-cases. To partially address the absence of information on diabetes before 1996, we also classified individuals in our sample as diabetics if they had fasting serum glucose levels ≥ 7.0 mmol/L.Information on diabetes in the AMORIS cohort was based on the National Diabetes Register51. In brief, these sub-cohorts contained data on 269,365 participants, ranging from 19 to 96 years of age at baseline (between 1974 and 2005) without a known tumour, after exclusion of 2,711 non-fasting participants. Data included measured height, weight and blood pressure, as well as levels of fasting plasma glucose, triglycerides and total cholesterol. For this study, each individual’s baseline data were taken from the first (or only) health examination with complete or near-complete data. Nationwide cancer and cause-of-death registries were used to follow participants. Glioma diagnoses were based on PAD codes 475 (n = 21) and 476 (n = 96) and ICD-7 193 codes in the Swedish sub-cohorts. In the Austrian sub-cohort these diagnoses were identified using ICD-7 193 and histology codes 93803, 93813, 93823, 94003, 94013, 94113, 94203, 94213, 94403, 94503, and 94513 from ICD-O1 . Here the exposure categories are different to allow comparison with the AMORIS cohort. More importantly, we evaluated time and age interactions and competing risks, which were not examined previously. Also, an exploratory analysis was performed using a subgroup with repeated exposure measurements.The association between blood glucose and glioma risk has been previously reported using all seven population-based Me-Can sub-cohortsIn our multivariable time-dependent Cox regression models, study participants were included in the analysis at their age at the time of their first blood glucose test (left-truncation) and exited the analysis at the age they were diagnosed with glioma or died, emigrated or when follow-up ended (whichever event came first). In addition, in the Me-Can cohort participants were also censored at the time of diagnosis of any malignant tumour. Follow-up time was restricted to a maximum of 15 years from the initial blood glucose test because we were interested in the hypothetical effect of the preclinical tumour on glucose levels  as well as the possible effects of blood glucose levels on glioma risk.18, the lowest cut point for the fourth glucose level (5.6 mmol/L) is, according to the American Diabetes Association54, the lowest level of blood glucose that indicates pre-diabetes, while the lower cut point for the fifth glucose level (6.1 mmol/L) is the lowest blood glucose value designated by the World Health Organization as an indication of impaired fasting glucose55. We used these cut points rather than quintiles because of their clinical significance.In the initial glucose dose-response analysis the first three lower cut points for the blood glucose variable were based on those in a previous paper using AMORIS cohort dataP = 0.03) and age at glioma diagnosis . We therefore used the strata statement in PROC PHREG  and stratified these regression models by both time before diagnosis and age at diagnosis thus producing separate baseline hazards for each time and age-specific stratum56. For consistency, the Me-Can data were also stratified by sub-cohort, but not adjusted for fasting status since the few non-fasting participants were not included in the data set. We tested dose-response trends over time and age using linear contrasts, analysis of variance, and F-tests57.Our time- dependent Cox regression models were adjusted for age at the time of the first blood glucose test, date at the time of the blood glucose test, fasting status, triglycerides, cholesterol levels, and gender. Using data from the AMORIS cohort, we found that when characterised by a single interaction term and stratified on the other time-related variable, the association between blood glucose and glioma varied with the time of the blood draw before glioma diagnosis  and four categories of age in years at glioma diagnosis . To avoid multicollinearity, we used two separate time-dependent Cox regression models; one representing time interactions and a second representing age interactions. To address the potential problem of the influence of outliers on the results, we used natural logarithms of the blood glucose values and standardised them to a mean of zero and a standard deviation of one. In addition, we accounted for that time-related variable which was not included in the model using the strata statement in PROC PHREG .All Me-Can cohort study participants fasted before their blood was drawn; however only 56% of the AMORIS study participants did so. We therefore analysed AMORIS glucose dose-response relationships separately by fasting status. We found that fasting and non-fasting glucose dose-response relationships were similar . Hazard ratios based on the latter model are those which would have been found had individuals who were at risk of glioma when they died remained in the population but were not subsequently diagnosed with gliomaAll analyses were conducted using SAS statistical software, version 9.4 .Supplemental Tables