Pain control during and after any surgical procedure, is extremely essential for the comfort of patients. Pain killers used routinely act by inhibiting cyclooxygenase to control pain and inflammation. Cox-1 is constitutively expressed in most cell types, including platelets, whereas Cox-2 is absent from most healthy tissues but is induced by pro-inflammatory or proliferative stimuli. Cox-1 plays a role in the production of prostaglandins involved in protection of the gastric mucosal layer and thromboxanes (TX) in platelets. Cox-2 generally mediates elevations of prostaglandins associated with inflammation, pain, and pyresis. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen are generally nonselective inhibitors of Coxs. This lack of selectivity has been linked to their propensity to cause gastrointestinal side effects. The new Cox-2 selective inhibitors, or coxibs, show the same anti-inflammatory, analgesic, and antipyretic effects as nonselective NSAIDs but are supposed to have reduced side-effect profiles. This study evaluates whether rofecoxib (50 mg) given one hour pre-operatively or the same drug given one hour post-operatively is more effective in controlling the pain and swelling in mandibular third molar surgery. Excellent pain control in dentistry is essential for the delivery of optimal dental care and for the well being of the patient. It is now known that high levels of cyclooxygenase -2 (Cox–2) are expressed at the sites of tissue inflammation leading to the synthesis of prostaglandins that mediate pain and inflammation.Prostaglandin synthesis in humans is catalyzed by two distinct isoforms of cyclooxygenase (Cox-1 and Cox-2). Cox-1 is constitutively active throughout the body, whereas Cox-2 expression is markedly upregulated by a variety of inflammatory mediators. These distinct expression patterns have led to the proposal that prostaglandins produced by Cox-1 are largely responsible for physiological functions, while Cox-2 derived prostaglandin mediate pathophysiological and inflammatory processes including pain.Cox-2 inhibitors like rofecoxib act by specifically blocking Cox-2 activities without blocking the other isoform of cyclo-oxygenase, namely Cox-1, which is responsible for gastric mucosal protection and vascular homeostasis. So we did a comparative study on the efficacy of rofecoxib, a Cox-2 inhibitor by administering the drug preoperatively for one group of patients and postoperatively for the other group.The study group comprised of those patients who came to the department of oral and maxillofacial surgery, Mahatma Gandhi Dental College and Hospital, Pondicherry for the surgical removal of impacted mandibular third molar. A total of 38 patients, that is, 19 patients in each group were included in the study. A randomization table was prepared manually and a randomly allocated number was given to each patient using this table. A proper clinical and radiologic evaluation was done preoperatively. The patients underwent the treatment under local anesthesia .The inclusion criteria were as follows:1.All types of impaction were taken up for the study.2.Patients of age 18 – 35 were included.3.Both sexes were included.4.Only patients willing to give an informed consent were made to participate in the study.The exclusion criteria were as follows:1.Hypersensitivity to analgesics.2.Patients who have taken tricyclic antidepressants, sedatives, analgesics, antibiotics, antihistamines, or corticosteroids in the last one month.3.Medically compromised patients.4.Pregnant ladiesThe two groups in the study were:Rofecoxib 50 mg orally, once daily and it was given 1 hour pre-operatively. A placebo was given 5 minutes after the surgery.For the patients in this group, the placebo was given one hour preoperatively. rofecoxib 50 mg orally, once daily was given five minutes post operatively.The patients in both the groups continued taking rofecoxib 50 mg (once daily) for another five days.In this triple blind study, the drugs were given to the patients by a post graduate teacher/staff nurse in the department who was neither the operator nor the observer.The following details were recorded preoperatively:Name of the patient, age, sex, address, date, randomization number, informed consent, tooth number, operator’s name and experience, type of impaction, and split/elevated.After giving inferior alveolar nerve block using 2% lignocaine hydrochloride containing 1:80,000 adrenalines, ward’s incision was placed in relation to the impacted third molar. The flap was raised and the tooth was removed either by splitting or elevation after bone removal. The wound was closed with 3-0 black braided suture.The surgical procedure was done by post graduates in the department. The operators were requested not to prescribe any pain killer or other drugs after the surgery. The operators were unaware of which drug was given preoperatively and post operatively.The patients were asked to assess their pain intensity at 5, 7, and 24 hours after surgery. The subjects were asked to record their pain Intensity (PI) on a 100 mm visual analog scale (VAS) by placing a mark on the line to indicate the magnitude of their pain.The subjects were asked to assess their pain intensity on a four-point categorical scale in the graph drawn above. The scoring scale was as follows:Score 0 – No pain.Score 1 – Mild pain.Score 2 – Moderate pain.Score 3 – Severe Pain.The patients were recalled 48 hours after the administration of the drug and they were asked about the pain relieving effect of the drug by a single observer who was unaware of the drug given. The graph was also seen by the observer.However, patients were advised to take a rescue medication (one Paracetamol tablet) if pain developed before the time at which rofecoxib was supposed to be taken. If the patient had taken the rescue medication, the pain intensity (PI) just before taking the rescue medication had to be marked on the graph by the patient himself/herself.The following details were recorded postoperatively:1.Duration taken for surgery.2.Pain intensity, after 5, 7, and 24 hours as marked in the visual analog scale.3.Whether patient has taken rescue medication.4.CRP test was done after 48 hours to assess the inflammatory response.C-reactive protein is one of the family of acute phase proteins found in the blood of both humans and animals. It is found in concentrations of less than or equal to 5 µg/ml in the serum of healthy persons. However, during an inflammation or infection, the levels may increase by as much as one thousand fold and can be detected as early as 5–10 hours after tissue damage. The increase in CRP levels in serum appears to be a non specific phenomenon but the change can be used to monitor the course of certain diseases and their treatment.Elevated level of CRP can be demonstrated in cases of acute myocardial infarction, rheumatoid arthritis, bacterial and viral infections, acute rheumatic fever, and several types of malignancies.In our study, we have assessed the CRP level of the patients both qualitatively and semi quantitatively using the ‘SPAN’ CRP test kit.Analgesics are routinely used after dental and oral surgical procedures. But the NSAIDs which are usually prescribed, though effective in controlling inflammation, have some undesirable side effects. So, we decided to evaluate the efficacy of coxibs (rofecoxib 50 mg) in controlling the inflammation after removal of impacted mandibular third molar by comparing two groups – one receiving the tablet 1 hour preoperatively and the other receiving the tablet 5 minutes postoperatively. The patients in both groups continued taking the tablet for a total period of 5 days.The mean age of patients in our study was 26.5 years, ranging from 18 years to 35 years. The number of males and females were 15 and 23, respectively, with a ratio of 1:1.5. For 31 patients the tooth was removed by splitting and for the other seven patients the tooth was removed by elevation. The type of impaction was mesioangular in 27 cases, distoangular in 10 cases, and horizontal in one case.The patients reported for follow-up after 48 hours and then after 1 week following surgery. One patient had numbness in the lower lip on the operated side and six patients had postoperative infection. These complications were managed conservatively.The maximum pain score as assessed using the visual analog scale was ‘1’ in the postoperative group and ‘2’ in the preoperative group, twenty four hours after surgery. The C-reactive protein level 48 hours after surgery ranged from <6µg/ml-96 µg/ml in the postoperative group and <6 µg/ml-48 µg/ml in the preoperative group.Two patients in the postoperative group and four patients in the preoperative group had taken rescue medication once after surgery. One patient in each of the group had taken rescue medication twice after surgery.A comparison was done between the two groups based on CRP level and the number of patients who required rescue medication. There was no statistically significant difference between both the groups in the level of inflammation after surgery .Development of the coxibs, a new group of anti-inflammatory drugs, represents a response to the unsatisfactory therapeutic profile of NSAIDs. Although NSAIDs are the most commonly used analgesic agents in ambulatory patients, their long term use is limited by gastrointestinal effects such as dyspepsia and abdominal pain and, less often, gastric or duodenal perforation or bleeding.In the recent past, the analgesic efficacy of coxibs had been compared with NSAIDs in the dental pain model. Malmstrom K., et al., compared the analgesic efficacy of rofecoxib with celecoxib and ibuprofen in patients experiencing pain after third molar surgery. They found that rofecoxib had superior analgesic effect and longer duration of action than celecoxib and ibuprofen, respectively.In our study we had assessed the efficacy of the cox-2 inhibitor rofecoxib based on the timing of its administration unlike most of the studies which had either compared its efficacy with another cox-2 inhibitor or a NSAID.Swan et al., by comparing the effects on renal function in patients receiving rofecoxib, indomethacin, and placebo concluded that rofecoxib has effects on renal function quite similar to nonselective NSAIDsThe ‘CLASS’ trial which lasted for 13 months concluded that there was no statistically significant difference between celecoxib and NSAIDs like ibuprofen and diclofenac in the incidence of upper gastrointestinal bleeding or gastric perforation.However, Claire Bombardier established a statistically significant difference in the upper gastrointestinal events by comparing rofecoxib with naproxen in patients with rheumatoid arthritis.Francesca Catella – Lawson had demonstrated that the concomitant administration of rofecoxib did not affect the pharmacodynamics of aspirin in patients with both arthritis and vascular diseases. However, we have excluded medically compromised patients from our study group.Muhammad. Mamdani did an observational cohort study over a period of 1 year to compare the rates of upper gastrointestinal hemorrhage in patients treated for arthritis with either cox-2 inhibitors or NSAIDs and concluded that there is a lower short term risk of upper gastrointestinal hemorrhage for selective cox-2 inhibitors compared with non-selective NSAIDs.Apart from control of inflammation and pain associated with it, the antiangiogenic activity of cox-2 inhibitors provides an additional rationale for the use of cox-2 inhibitors in severe painful conditions like rheumatoid arthritis. James. M. Woods demonstrated that rofecoxib acts directly on human dermal microvascular endothelial cells and inhibit their chemotactic and tube forming ability.The cox-2 inhibitors are believed to be associated with an increased risk of myocardial infarction and congestive cardiac failure. ,34 Hyon Muhammad Mamdani (2004)  suggesteGizzarelli compared the efficacy of preoperative administration of rofecoxib with ibuprofen in third molar extraction. The results showed that at 6 hours rofecoxib group had significantly better pain relief than ibuprofen group.Kyle.S. Christensen in a randomized placebo-controlled trial on patients experiencing moderate or severe pain within 4 hours after multiple third molar extractions showed that valdecoxib had a significantly faster onset of action and superior analgesic effect than rofecoxib.There are studies ‘for’ and ‘against’ the use of cox-2 inhibitors as an alternative to NSAIDs in patients who develop adverse drug reaction to NSAIDs. However, in our study we had not been confronted with a situation in which an allergic reaction to rofecoxib occurred.This study group comprised of those patients who came to the department of oral and maxillofacial surgery, Mahatma Gandhi Dental College and Hospital, Pondicherry for the surgical removal of impacted mandibular third molar. The study group comprised of one group for which rofecoxib 50 mg was given one hour preoperatively and the other for which the same drug was given 5 minutes postoperatively.Five patients in the preoperative group and three patients in the postoperative group required rescue medication despite taking rofecoxib 50 mg once daily for five days including the day of surgery. No significant statistical difference was observed in the CRP level between both the groups.Our study concluded that the timing of administration of drug (preoperatively or postoperatively) does not have any significant difference in controlling the pain and swelling in mandibular third molar surgery.The authors confirm that there are no conflicts of interest.


LEAFY (LFY) and the F-box family member UNUSUAL FLORAL ORGANS (UFO) have been found to promote floral meristem identity across diverse dicot model systems. The lower eudicot model Aquilegia produces cymose inflorescences that are independently evolved from the well-studied cymose models Petunia and tomato. We have previously characterized the expression pattern of the Aquilegia homolog AqLFY but in the current study, we add expression data on the two UFO homologs, AqUFO1 and 2, and conduct virus-induced gene silencing of all the loci. Down-regulation of AqLFY or AqUFO1 and 2 does not eliminate floral meristem identity but, instead, causes the transition from inflorescence to floral identity to become gradual rather than discrete. Inflorescences in down-regulated plants generate several nodes of bract/sepal chimeras and, once floral development does commence, flowers initiate several whorls of sepals before finally producing the wildtype floral whorls. In addition, silencing of AqUFO1/2 appears to specifically impact petal identity and/or the initiation of petal and stamen whorls. In general, however, there is no evidence for an essential role of AqLFY or AqUFO1/2 in transcriptional activation of the B or C gene homologs. These findings highlight differences between deeply divergent dicot lineages in the functional conservation of the floral meristem identity program.Homologs of the transcription factor  Arabidopsis thaliana and Antirrhinum majus revealed homologous genes that control IM and FM identity: IM identity being primarily promoted by orthologs of the A. thaliana gene TERMINAL FLOWER  and the ALOG family member TERMINATING FLOWER  and two AGAMOUS (AG) homologs, as well as single copies of PISTILLATA (PI) and FRUITFULL-like (FUL), the closet relative to AP1 , and germinated and grown under long day  at 18°C.Seeds for AqLFY has been previously identified (Aquilegia coerulea (James) genome available on Phytozome v.12.1 (http://www.phytozome.net/). To identify AquilegiaUFO homologs, we conducted a BLAST search with default settings using the Arabidopsis UFO sequence as the query. Together with previously identified UFO orthologs and closely related F-box family members (http://tree.bio.ed.ac.uk/software/figtree/) and color prepared using Adobe Illustrator CC 2015. These analyses led to the assignment of gene names to each locus: AqUFO1 (Aqcoe1G161900) and AqUFO2 (Aqcoe4G199100). We should also note that there appears to be an almost identical copy of AqUFO2 on an unassembled scaffold (Aqcoe0095s0001), but the predicted transcript differs by only a six nucleotide insertion in a repetitive stretch of the 3’ end of the coding region, making it extremely difficult to distinguish between them. For the purposes of this study, we are considering Aqcoe4G199100 and Aqcoe0095s0001 to be equivalent.entified  but was  members , amino a members  as impleAqUFO1 (383 bp) and AqUFO2 (328 bp) from non-conserved regions of the open reading frame were PCR amplified using primers listed in ® vector. Both sense and anti-sense probes of each gene were alkaline hydrolyzed to an average length of 150 bp. All in situ hybridization steps were performed as described by Fragments of Aquilegia VIGS protocol and construction of the TRV2-AqANS positive control plasmid has been described previously  as soon as they were removed from cold treatment. After multiple failed silencing attempts using the standard protocol, we found that this short vernalization treatment was necessary to achieve the early inflorescence silencing needed to affect AqLFY and AqUFO1/2. Following the treatment, plants were returned to the long day conditions at 18˚C. Flowers showing any AqANS silencing were photo-documented and, upon maturation, the flowers were dissected and organs counted. The organ count datasets were subjected to ANOVA to detect differences between treatment classes followed by Scheffé’s test to detect pairwise difference between the classes, which have unequal sizes. All individual organs were photographed using a Kontron Elektronik ProgRes 3012 digital camera mounted on a Leica WILD M10 dissecting microscope . For every flower showing silencing, a selection of organs from each whorl was frozen at -80°C for subsequent RNA analysis.The eviously . To makeAqIPP2  was used for normalization as it has been previously shown to have little quantitative transcriptional variation across tissues and developmental time points (-ΔΔCT method described in Livak and Schmittgen (2001), taking into consideration the specific primer efficiencies as well as the exact fragment lengths.Total RNAs were prepared from collected floral buds and organs using the Qiagen RNeasy kit according to manufacturer’s instructions . Each RNA sample was treated with TURBO DNase  following manufacturer’s instructions. Individual RNAs were quantified using Nanodrop  and cDNAs were synthesized using 1 µg of total RNA . All primer sequences are listed in e points . Primer AqLFY, AqUFO1 and AqUFO2, we dissected six to eight stage 8-10 floral buds exhibiting multiple sepal whorls from silenced inflorescences. RNA was prepared from each meristem and tested as a separate bioreplicate. This was to address the fact that these loci are not expressed in mature floral organs. Comparable buds were dissected from AqANS-silenced inflorescences to serve as controls. The dissected mature organs from silenced flowers were assessed for expression of AqAP3-1, AqAP3-2,AqAP3-3, and AqPI. For each class of dissected organs, we analyzed three to five separate bioreplicates, and each bioreplicate was analyzed in three technical replicates. A two-tail Mann Whitney U Test was used to determine the statistical significance of differences between experimental and control values from AqANS-silenced plants. All results presented are the mean ± standard deviation of the examined samples, normalized to the appropriate reference samples as described above. All error bars represent standard deviationTo assay down-regulation of AqLFY, AqUFO1, and AqUFO2 were PCR-modified with primers containing 5’ and/or 3’ flanking EcoRI sites and cloned into pCR4-TOPO (Invitrogen). The subsequent inserts were cloned in-frame into pGBKT7 or pGADT7 vectors . Indicated Y2H protein pair combinations , previously shown to have strong protein–protein interactions in this system  than the AqANS-silenced controls, although there were no significant differences in the numbers of each of the other floral organs  in these aqlfy buds and found that both loci appeared to be up-regulated, especially AqUFO1. The B gene homolog expression levels were generally similar to control sepals in the additional sepal whorls, as well as in the petals , so we used in situ hybridization to determine the expression of these genes in developing floral meristems , aqufo2 , and aqufo1/2  in Petunia or tomato, strong loss of FM identity (very similar to strong lfy) or 2) in A. thaliana or A. majus, transformation of petals and stamens towards sepals or filaments, respectively, as well as variation in organ number and disorganized phyllotaxy .We used yeast two-hybrid to test the ability of AqLFY to interact with UFO homologs , the rice UFO ortholog, is quite broadly expressed, including in lateral organs, but its function appears to be largely unrelated, as well as novel, in that it delays the transition to FM identity rather than promoting it  in an auto-regulatory feedback loop that stabilizes their mutual expression . In Eschscholzia, the authors did see sepal/petal chimeras, but these appeared to represent a transformation grade from outer sepals to inner organs that had full petal identity. Therefore, while these phenotypes are consistent with a delay in activating B gene expression, once transcription is initiated, there is no evidence of a significant problem with AP3/PI expression.In the flower itself, one possible explanation for the additional sepal whorls is that they are connected to the initiation of B gene expression. Both reviewed . Consistreviewed , resultiAquilegia, it appears that the extra sepals are not due to a shift in homeotic gene expression but, rather, the intercalation of additional sepal whorls. In Eschscholzia, where Wreath et al. observed more widespread homeotic phenotypes and variation in organ number , it is less clear whether the additional calyx whorls were due to intercalation or homeosis. However, the presence in both taxa of internodal elongation between individual sepals or outer sepal whorls is a clear indicator of mixed meristem identity that supports this alternative interpretation. The observation of similar phenotypes in Aquilegia and Eschscholzia suggests that, across the Ranunulales, LFY and UFO homologs may primarily function to reinforce the switch from IM to FM identity in these cymose inflorescences, such that it is discrete and complete.An alternative explanation that could explain both the sepal/bract chimeras and the additional sepal whorls in the flower is a failure to definitively activate FM identity. It may be that these meristems are gradually transitioning through a phase of mixed IM/FM identity in which they first initiate lateral organs that have sepal morphology but have the elongated internodes typical of bracts. Then, even when they express enough FM identity to generate whorls of sepals, the meristems are getting “stuck” on first whorl production such that it takes several whorls before they can move on to the rest of the floral organs. Based on organ counts in AqLFY or AqUFO1/2 does not eliminate FM identity, but in their absence, the switch becomes sloppy and gradual. This is reminiscent of the feed-forward loop that appears to act downstream of LFY in Arabidopsis  are shared among all the cohorts, targeted silencing of the AqUFO loci had a much stronger impact on both the presence of petals and the number of stamens. Interpreting this phenotype has several challenges, including the variable nature of VIGS itself and the fact that AqLFY expression is also reduced in aqufo flowers. The implication would be that the phenotypic differences between the aqlfy and aqufo flowers reflect functions that are specific to AqUFO1/2 and independent of AqLFY. Based on what we know about UFO homolog function in other taxa, there are at least two potential contributing factors to these phenotypes.Another intriguing aspect of the observed phenotypes is the differences between the ufo mutants commonly exhibit petal-to-sepal and stamen-to-carpel homeosis, consistent with the role of UFO in activating transcription of the B class genes with LFY  the identity of petals and stamens, and 2) the initiation of second whorl organs  a loss of AqAP3-3 expression leading to petal-to-sepal transformation, and/or 2) the combined effects of a loss of FM identity that produces extra sepal whorls and the deletion of petal and outer stamen whorls.Second, we must also consider the reduction of stamen numbers in the l organs . In Aquiaqufo impacts stamen numbers in a manner that is not observed in aqlfy. In A. thaliana, the loss of petal initiation in alleles such as ufo-11 appears to depend on LFY function  initiation could be independent of AqLFY. It is interestingly to note in this regard that EscFLO silencing in Eschscholzia also results in reduced organ number in some flowers, particularly the stamens  and California State University, including the CSU-LSAMP program to DW and California State Polytechnic, Pomona to BS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


All procedures included the use of local anaesthesia (lidocaine). Place conditioning was tested 2 days after the last treatment by allowing calves to freely roam between the pens where they had previously been disbudded. Calves spent more time, and lay down more frequently, in the pen where they received meloxicam compared with the pen where they only received a local block. Surprisingly, calves avoided the pen where they received ketoprofen compared with the control treatment pen. We hypothesize that the shorter duration of action of ketoprofen resulted in increasing pain at the end of the conditioning period, explaining the increased aversion to this treatment. These results illustrate the value of place conditioning paradigms to assess the affective component of pain in animals, and suggest that the animal's evaluation of painful events depends upon the time course of when the pain is experienced.Pain in animals is typically assessed using reflexive and physiological responses. These measures allow inferences regarding nociception but provide little basis for conclusions about the affective component of pain (i.e. how negatively the experience is perceived). Calves routinely undergo painful procedures on commercial farms, including hot-iron disbudding, providing a convenient model to study pain in animals. The aim of this study was to investigate the affective component of post-procedural pain due to hot-iron disbudding, using conditioned place aversion. Calves ( This reProviding calves with a post-procedural analgesic, such as a nonsteroidal anti-inflammatory drug (NSAID), has been shown to reduce reflex and automatic pain responses ,15, but The main objective of the current study was to identify whether providing post-procedural analgesics could mitigate the affective component of pain in dairy calves following hot-iron disbudding. We used conditioned place aversion to compare two disbudding procedures: one without post-procedural pain control and one with the use of an NSAID. A secondary objective was to evaluate two different NSAIDs commonly used in veterinary practice to alleviate post-procedural pain: meloxicam and ketoprofen. We predicted that calves would display conditioned place aversion to the environment where they did not receive post-procedural pain control in comparison with the environment where they received either meloxicam or ketoprofen.2.(a)n = 34) were enrolled at 32 ± 5.9 days of age with a body weight of 68 ± 6.8 kg. Calves were assigned in blocks and balanced to two treatment groups: meloxicam and ketoprofen. See electronic supplementary material for details.Holstein heifers ((b)et al. [The experimental apparatus was identical to the one used in Ede et al. . Briefly(c)Calves were all given one session where they were provided free access to all three pens simultaneously within the apparatus before receiving treatments to avoid a potential effect of novelty.NSAID  and Control (where they did not). During treatments, calves were kept in the pens for 6 h. See electronic supplementary material for details.Calves were disbudded one bud at a time during two separate treatment sessions that took place 24 h (Treatment 1) and 72 h (Treatment 2) after pre-exposure. The two treatments were counterbalanced across the two treatment pens . Hence, all calves were subjected to both procedures: Testing occurred 48, 72 and 96 h after the second treatment . The test procedure was similar to pre-exposure: the calves were brought to the apparatus, where the removable gates had been withdrawn. During testing, calves were allowed free access to all three pens until they lay down  or after 60 min had passed; they were then returned to their home pen.(d)χ2 test. See electronic supplementary material for details.Time spent in each of the treatment pens (s) during testing was analysed with a mixed linear model using the lme4 R package ,17. The 3.meloxicam group and 15 in the ketoprofen group.Three calves were excluded from the study: two for not fulfilling pre-exposure criteria  and one for falling sick between the first and second treatment , which resulted in 16 calves in the t-test, t30 = −0.3, p = 0.7).During pre-exposure, calves did not differ in the time spent in the two different pens  but there was an interaction between treatment received in the pen (Control or NSAID) and the NSAID used (meloxicam or ketoprofen) . We, therefore, repeated the test of treatment separately for the two NSAIDs. In the meloxicam group, calves spent more time in the pen where they had received the NSAID compared with the control pen where no NSAID was provided . In the ketoprofen group, calves spent less time in the NSAID pen compared with the control pen .After conditioning, time spent in the pen was not affected by test session number, order of treatment, colour of treatment pen and side of the first bud disbudded , and calves in the ketoprofen group showed the opposite pattern .Out of the 93 tests , there were five in which calves did not lie down within the 60 min session . Among the remaining tests, calves in the meloxicam group lay down more frequently in the NSAID pen  compared with control calves that had not received a local block . There iKetoprofen can have a number of negative effects  when administered to humans –28. We dAs our experiment did not include a sham treatment in which calves were not disbudded, our results do not provide evidence that meloxicam eliminates the post-procedural pain associated with disbudding, only that it makes the memory of the procedure less aversive. We also note that the post-procedural pain associated with disbudding is thought to exceed the 6 h duration of focus in this study ,29,30. CAlthough we did not observe an effect of treatment order, it is possible that our sample size did not allow us to detect the impact of the lingering pain from the first treatment during the second disbudding. There are other factors worthy of attention regarding pain associated with horn removal, including the method used (e.g. amputation , caustic5.Meloxicam treatment made hot-iron disbudding less aversive to calves during the 6 h following the procedure, but ketoprofen treatment made the experience more aversive. Further research is needed to determine the number and timing of analgesic treatments required to control post-procedural pain when disbudding. We recommend the use of place conditioning to explore the affective component of pain in animals.


This study aims to determine whether 3 sessions per week of acupuncture treatment is superior to 1 session per week of acupuncture treatment for symptomatic outcomes in knee osteoarthritis.This is a two parallel-group, assessor-blinded, randomized controlled trial. Sixty patients with knee osteoarthritis (Kellgren–Lawrence grade II or III) will be recruited and randomly allocated to receive 24 or 8 sessions (group M or group L) of acupuncture treatment in a 1:1 ratio. Patients in group M will receive 3 sessions per week of acupuncture for 8 weeks. Patients in group L will receive acupuncture once per week for 8 weeks. The primary outcome is the response rate—the percentage of patients achieving a decrease ≥ 2 points on a numerical rating pain scale and a decrease ≥ 6 points in the Western Ontario and McMaster Universities Osteoarthritis Index function score at 8 weeks compared with baseline. Secondary outcomes include pain, function, overall effect, quality of life, and treatment credibility and expectancy.Three sessions per week of acupuncture treatment may be superior to 1 session per week of acupuncture treatment for symptomatic outcomes in knee osteoarthritis. Results of the study will be of great importance for the guidelines of clinical therapy.NCT03359603. Registered on 1 December 2017.Clinicaltrials.gov, The online version of this article (10.1186/s13063-019-3528-8) contains supplementary material, which is available to authorized users. Knee osteoarthritis is a common and disabling condition, particularly in people over 50 years of age . The estThere is currently no consensus on the best treatment to improve knee osteoarthritis symptoms. Pharmacologic modalities used to treat the symptoms of this disorder are associated with various side effects such as renal toxicity, serious gastrointestinal complications and cardiovascular events . In factNonpharmacological methods such as education and self-management, exercise and physical activity are recommended in the guidelines , 8. HoweAcupuncture is one of the most common complementary and alternative medical therapies and a popular treatment for pain and dysfunction associated with musculoskeletal conditions, and the effects of acupuncture are maintained over time , 13. In Based upon previous high-quality studies, we speculate that the clinical effectiveness of acupuncture for knee osteoarthritis may relate to treatment frequency. The treatment frequency in acupuncture trials with knee osteoarthritis which showed negative results , 18 is sThis is a pragmatic, parallel, two-armed randomized controlled exploratory study with 8-week follow-up. The protocol has been registered on ClinicalTrials.gov (No NCT03359603) and will be conducted in accordance with the Declaration of Helsinki. The study will be reported in accordance with the Consolidated Standards of Reporting Trials (CONSORT) statement and its relevant extensions to randomized pilot and feasibility trials . The proThe recruitment will be conducted at Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University. The study plans to recruit 60 patients with knee osteoarthritis using acupuncture students to do the recruiting. The study was approved by the medical ethical review committee of Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University (No. 2017BL-076-01). Patients will be recruited through WeChat , outpatient units and print advertisements. People willing to participate in the study will be invited to contact the clinical research coordinator (CRC) by telephone. The CRC will perform a preliminary screening of people for inclusion and exclusion criteria, and then, if appropriate, schedule a face-to-face baseline visit with a senior specialist in physical and rehabilitation medicine. Patients will also be referred for radiographic evaluation. All eligible patients will provide written informed consent  criteria .Symptoms have been present for more than 6 months.Radiologic confirmation of knee osteoarthritis  .Agreed to refrain from the use of any analgesics during the trial (paracetamol sustained-release tablets will be given to patients if their pain intensity reaches ≥ 8 out of 10 on an 11-point NRS).Signed informed consent.History of knee surgery or waiting for surgery (knee replacement or knee arthroscopy).Knee pain caused by other diseases .History of arthroscopy within 1 year or intra-articular injection within 4 months.History of receiving acupuncture treatment within 3 months.Severe acute/chronic organic or mental diseases.Pregnant and lactating women.Coagulation disorders .Participation in another clinical study in the past 3 months.History of a cardiac pacemaker, metal allergy or needle phobia.Patients will be randomly assigned (on a 1:1 ratio) to the more frequent acupuncture treatment group (group M) or the less frequent acupuncture treatment group (group L). The randomization sequence will be computer generated by independent research staff with SAS 9.3 software, using block randomization with a block size of six via a central randomization system. The randomization sequence will be embedded into the software . The CRC will input the patient information on a tablet computer and will be given a random number. The randomization protocol will be designed by a statistician (Y. Wang) of Fuwai Hospital, China Academy of Medical Science who is not involved in the later statistical work of the study.The outcome assessor and data analyst will be blinded to group assignments. The acupuncturist and patients will not be blinded because of the nature of the intervention. Patients’ allocated interventions will not be revealed until the statistical analysis is completed.Bi syndrome. Furthermore, the therapy was discussed with experts in the field of acupuncture.The treatment strategies for acupuncture are generally considered a pragmatic compromise between the need for some standardization and the need for individualization. This treatment is based on a distinguished Chinese textbook, as well as traditional Chinese medicine meridian theory to treat The most important local points in the literature were chosen as obligatory points. Therefore, the following five local points have to be used for every treatment session on the affected knee: ST 35, EX-LE5 (Neixiyan), LR 8, GB 33 and an Ahshi point. Another three customized acupoints should be chosen for therapy in accordance with the individual localization of the meridians that traverse the area of the most painful point, with the intent of optimizing efficacy. ST34, ST36, ST32, ST40 and EX-LE2 will be used for pain at the stomach meridian. GB31, GB36, GB34, GB39 and GB41 will be used for pain at the gallbladder meridian. BL39, BL40, BL57 and BL60 will be used for pain at the bladder meridian. LR7, SP9, SP10, KI10, SP4, SP6, LR3 and KI3 will be used for pain at the three-yin meridians of the foot . The depth of needling should be 10–30 mm, depending on the point location. Needles will be stimulated manually for 10 s to achieve “De Qi”. Paired alligator clips will be attached transversely to the needle holders at LR 8-GB 33 and two other customized acupoints. An electric stimulator  will be applied to the four acupoints. The electro-acupuncture stimulation will last for 30 min with a dilatational wave of 2/100 Hz and a current intensity (0.1–1.0 mA) depending on the patient’s comfort level (preferably with skin around the acupoints shivering mildly without pain). Other needles will be manipulated for 10 s during the treatments. If both knees are affected, both knees will be treated with acupuncture as indicated. However, the electric stimulator will be used on only one knee (more painful).The acupuncturist will be required to have a Chinese medicine practitioner license and have been qualified for at least 10 years. Paracetamol sustained-release tablets  will be given to patients if their pain intensity is ≥ 8 out of 10 on an 11-point NRS. Acupuncture treatment will be discontinued if the patients suffer from any adverse events and the acupuncture physicians can decide to terminate the trial.Thirty patients in group M will receive acupuncture treatment of 3 sessions per week  for 8 weeks. Patients will not be allowed to take any analgesic or anti-inflammatory medications during the trial. In cases of intolerable knee pain, the patients will be instructed to take Tylenol as a rescue medication. The usage of Tylenol will be documented by the outcome assessors. The use of other treatments, such as injections of any kind, moxibustion, ear acupuncture or cupping, will not be allowed.Thirty patients in group L will receive acupuncture treatment once per week  for 8 weeks. Other interventions will be similar with group M.If only one knee is affected, the assessment of the outcomes will relate to this knee. If the patient has two affected knees of which only one meets the ACR criterion and Kellgren–Lawrence grade II or III, only this knee will be evaluated. In the case that both knees are affected in accordance with the inclusion criteria (ACR and Kellgren–Lawrence grade II or III), the more painful knee will be randomly chosen for evaluation.The response rate is the percentage of patients with improvement in average pain (NRS) of at least 2 units and in the Western Ontario and McMaster Universities Osteoarthritis Index  (WOMAC) Knee pain will be assessed by the NRS and the WOMAC pain subscale  at weeks 4, 8 and 16. The WOMAC function subscale will be used to measure physical function at weeks 4, 8 and 16. The WOMAC stiffness subscale will be used to measure the degree of stiffness experienced in the knee at weeks 4, 8 and 16. The Overall Treatment Effect will be used to measure the overall acupuncture treatment effect at weeks 4 and 8. The 12-item Short Form Health Survey   will be completed on paper copies and then entered into the Excel spreadsheet by an independent investigator to act as the first level of control to ensure the accuracy of the data. The second level of data integrity will include data monitoring and validation that will be conducted on a regular basis throughout the study. The original CRFs and all other forms (including the consent forms) will be archived securely at the School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine. The Research Ethical Committee of Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University will audit the trial conduct every 5 months independently of investigators and the sponsor, and will decide on any premature closure of the study.The current study is designed as a pilot study to explore symptomatic improvement in patients with knee osteoarthritis receiving 3 sessions per week of acupuncture compared to 1 session per week. Acupuncture as a complex intervention is different from drugs. If the acupoints in the study were changed, then the effectiveness of the acupuncture would be altered. Consequently, we do not use data from the literature to calculate the sample size. We based our sample size on the response rate. Based on our previous study (unpublished) and clinical experience, the sample size is calculated to provide 80% power if 70% of group M achieved a decrease ≥ 2 points on the NRS and a decrease ≥ 6 points in WOMAC function score at 8 weeks, compared with 30% of group L, at a two-tailed α level of 0.05. This requires a total of 60 participants, allowing for 20% dropout.2 test. For secondary outcomes, continuous variables including WOMAC pain, function and stiffness scores, NRS, SF-12, the Expectation and Credibility of acupuncture treatment and Overall Treatment Effect will be compared between the two groups at all follow-up time points using an unpaired Student’s t test or Wilcoxon rank-sum test as appropriate. For the dropout analysis, we will use the last observation carried forward method. SPSS version 21.0  will be used for statistical analysis.Analyses will be intention-to-treat (ITT), with all patients included in their assigned treatment group and receiving at least one session treatment. The level of significance will be set at 0.05 (two-sided). Patients’ baseline characteristics will be summarized by treatment arm. For continuous outcomes, the data will be presented as mean (standard deviation) or the median (interquartile range) according to the normality of the distribution. Enumeration data will be presented as a percentage. For the primary outcome, we will calculate the response rates at 8 weeks and compare group M and group L using the χEthics approval was obtained from the Research Ethical Committee of Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University (No. 2017BL-076-01). This trial has also been registered on ClinicalTrials.gov (No. NCT03359603) and will be reported in compliance with the CONSORT statement as well as Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA) . PatientThis study is designed to explore an effective acupuncture method for knee osteoarthritis.We chose a frequency of 3 sessions per week as one kind of electro-acupuncture intervention, because 3 sessions per week is most commonly used in clinical practice in China to treat chronic pain. We chose a frequency of once per week as another intervention, because electro-acupuncture treatment once per week was usually used in previous studies. The study is a single-site randomized clinical trial. A single-site trial might be helpful in the aspect that the quality of the study procedure will be better controlled in a single site than in multiple sites. One potential limitation of this study is that the unblinded treatment might theoretically influence reporting or measurement of the outcomes, thus introducing bias. Therefore, we will use an Expectation and Credibility of treatment rating scale to measure the patients’ attitudes to acupuncture.The information obtained from the exploratory study will inform the decision to conduct a full-scale randomized controlled trial to test the frequency dependence of acupuncture, which will add to the current small body of high-quality evidence that is available on the optimal number of sessions of acupuncture for the management of knee osteoarthritis.This trial is currently recruiting patients.Additional file 1:Completed Standard Protocol Items: Recommendation for Interventional Trials (SPIRIT) 2013 Checklist: items addressed in this clinical trial protocol. (DOC 115 kb)Additional file 2:Model consent form (DOCX 26 kb)


Novel redox mediators and HTMs are key elements to reach higher performing DSC as they can offer much higher output voltage than the traditional triiodide/iodide redox couple.Dye-sensitized solar cells have been investigated intensively during the last three decades. Nevertheless, there are still many aspects to be explored to further improve their performance. Dye molecules can be modified endlessly for better performance. For instance, steric groups can be introduced to slow down recombination reactions and avoid unfavorable aggregation. There is a need for more optimal dye packing on the mesoporous TiO Efficient dye-sensitized solar cells (DSCs) were first developed in the 1990s marked by the breakthrough work by O'Regan and Grätzel  who firs2 working electrode (WE), a redox electrolyte and a counter electrode (CE). Both the WE and CE can be (semi) transparent, which allows for illumination of the solar cell from either side. Dye molecules, equipped with suitable anchoring groups, are adsorbed as a monolayer onto the mesoporous TiO2 electrode. When the dyes absorb light, the excited molecules can inject electrons into the conduction band of TiO2 (electron transfer (ET) reaction 1 in 2 are collected at the underlying fluorine-doped tinoxide (FTO)-coated glass substrate and move through an external circuit to the counter electrode, where they reduce the oxidized redox mediator (ET 3), thus completing the cycle. A schematic presentation of these processes is shown in The working mechanism of DSC differs much from other types of solar cells  or to the oxidized form of the redox mediator (ET 5). For optimized DSC systems the incident photon to current conversion efficiency (IPCE) is about 90%. Since there are some reflection and transmission losses, this implies that every absorbed photon gives an electron in the external circuit under the measurement conditions used, typically under short-circuit conditions. Under operating conditions, when the solar cell delivers maximum power output , the IPCE is significantly lower and there are both current and voltage losses. In order to fully optimize the DSC these losses must be minimized, as will be discussed in section Limiting Factors in the DSC.A number of recombination reactions in the DSC compete with the forward processes described above. Radiative and non-radiative de-excitation of the dye competes with electron injection from the excited dye into the conduction band of TiO−) redox couple is most frequently used in DSCs. However, this complex redox couple has some serious limitation, as pointed out in our previous work  is relatively negative (+0.35 V vs. NHE) compared to the E0' (D+/D) of typical dyes (located at ca. 1 V vs. NHE), resulting in a large loss of more than 0.5 V. The origin lies in the multistep regeneration mechanism that involves formation of the The triiodide/iodide  limit gives the maximum efficiency attainable for a single junction photovoltaic device, which is 33.8% under 1,000 W mn Green, . While tVOC) in semiconductor photovoltaics has Eg/e as the absolute upper limit, for DSC the upper limit is set by the difference between the conduction band potential EC of the TiO2 on one side and the redox potential of the electrolyte on the other side. This value will always be smaller than the “bandgap” of the dye. EC of TiO2 anatase is about −0.5 V vs. NHE when its surface is uncharged . This level can be altered by additives in the electrolyte that lead to a change in the surface charge or alter the dipole moment at the semiconductor/electrolyte interface. Alternatively, EC can be changed by chemical modification of the TiO2, for instance by incorporation of Mg in the structure, which can to shift EC to more negative potential, to about −0.7 V vs. NHE  covering the mesoporous TiO2 can also affect the location of EC , nc is the density of conduction band electrons, and Nc is the effective density of electronic states at the bottom of the conduction band. Nc is a material constant and is about 1020 cm−3 for TiO2 anatase. Under illumination at open-circuit conditions EF depends on nc, which depends in turn on the generation flux of injected electrons and the rate constants for electron recombination. As a rule of thumb, EF is about 0.1 V more positive than EC at 1 sun.where, − redox couple, which has very favorable electron transfer kinetics, giving very low recombination losses and high JSC. This comes, however, at the cost of a relatively low VOC due to the rather negative value of the redox potential. Successful alternative redox mediators have more positive redox potential and can provide higher VOC. Nearly all of these redox mediators give, however, faster electron recombination to oxidized redox mediators, thereby lowering the Fermi level on the TiO2 under operational conditions.Formal reduction potentials of a series of redox mediators for DSC are listed in VOC for a DSC is 1.4 V, and was obtained for a Mg-doped TiO2 with additional surface modification by MgO and Al2O3, sensitized by a coumarin dye, and in combination with the Br− redox couple 3-DSC.The best performing DSCs with Co(bpy)* should overlap with acceptor levels in the conduction band of TiO2 for efficient electron injection. A lower value of λ would allow for closer matching between EC of the semiconductor and the calculated standard potential for the excited dye, E0(D+/D*) and less voltage loss in the DSC. The reorganization energy of the dye is due to internal molecular reorganization of the dye when it changes redox state and external reorganization of the solvent shell. Due to the absence of solvent, reorganization energies should be lower in solid-state DSC.As indicated in 2, which will accelerate the recombination process significantly  the electron transport in the mesoporous TiO2 is not a limiting factor, since transport becomes faster upon more electron accumulation, resulting in a more negative potential for EF. In other words, the transport resistance Rtr becomes smaller when the TiO2/electrolyte capacitor CTiO2 is charged, see A long electron lifetime is favorable for the DSC as it will improve the Rext is infinite. All current then goes through the recombination resistance Rrec. This is the normal condition to measure electron lifetime τ, which is equal to Rrec × CTiO2. Under short circuit conditions Rext is equal to 0. The charge collection efficiency : ηCC is then given by Rrec/(Rseries + Rtr + Rrec), where Rseries is the series resistance discussed below. Under MPP-conditions we find: ηCC = Rrec /(Rseries + Rtr + Rext + Rrec).Under open-circuit conditions the external resistor Based on this simple scheme, it is evident that the recombination resistance must be maximized to increase current collection efficiency and output voltage across the external resistor under MPP conditions. The recombination resistance will decrease with increasing thickness and the surface area of the mesoporous electrode. Increasing thickness will, however, increase the generated photocurrent. For every specific DSC system there is an optimum film thickness: Typical values are ~10 μm for regular liquid electrolyte DSC and ~2 μm for solid-state DSCs.Rseries down to 1.8 Ohm cm−2 by optimizing the catalytic performance of the counter electrode and the electrolyte distance between working and counter electrode  is by far the most used wide-bandgap semiconductor electrode used in DSC, and so far the most successful. In several studies the nanoparticle size, film porosity, and TiCl4 after treatment  a much improved voltage can be obtained . In general, other metal oxides can function a mesoporous electrode in DSC devices, but so far their performance is lower than that of their TiO2 counterparts.ZnO has been studied intensively as a nanostructured electrode in a wide variety of morphologies in the DSC  with an organic dye possessing a pyridine binding group . Most promising results have been achieved with molecular organic hole conductors such as spiro-MeOTAD, conduction polymers such as PEDOT, and very recently, with metal complexes 2, LiTFSI, and perhaps 4-tert butylpyridine needs to be explored.The surprising finding that dried-out DSC with Cu-complex redox electrolyte were still functioning as efficient solar cells, led to new type of ssDSC, the so-called zombie solar cell (Freitag et al., 2/dye interface.In general, ssDSC are highly attractive for practical application, but their performance needs to be improved. The introduction of an intermediate redox level may be helpful: it could remove the formed holes rapidly away from the TiO2 surface to increase light absorption and to achieve a better blocking effect. Co-sensitization offers good possibilities in this respect. Novel redox mediators and HTMs are key to higher performing DSC as they can offer much higher output voltage than the traditional triiodide/iodide redox couple. High-performance DSC are of interest for many applications, ranging from power source for consumer electronics, to building integrated PV and large-scale power generation. The option of high transparency in the near-infrared region also opens up for the use of DSC as a top cell in tandem solar cells devices.Despite three decades of intense research on dye-sensitized solar cells, there are still many aspects to be explored to further improve their performance. Nearly infinite types of modifications of dye molecules are possible, where steric groups can be introduced to slow down recombination reactions. There is a need for more optimal dye packing on the TiOThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Patients hospitalized with heart failure suffer the highest rates of 30-day readmission among other clinically defined patient populations in the United States. Investigation into the predictability of 30-day readmissions can lead to clinical decision support tools and targeted interventions that can help care providers to improve individual patient care and reduce readmission risk.This study aimed to develop a dynamic readmission risk prediction model that yields daily predictions for patients hospitalized with heart failure toward identifying risk trajectories over time and identifying clinical predictors associated with different patterns in readmission risk trajectories.A two-stage predictive modeling approach combining logistic and beta regression was applied to electronic health record data accumulated daily to predict 30-day readmission for 534 hospital encounters of patients with heart failure over 2750 patient days. Unsupervised clustering was performed on predictions to uncover time-dependent trends in readmission risk over the patient’s hospital stay. We used data collected between September 1, 2013, and August 31, 2015, from a community hospital in Maryland (United States) for patients with a primary diagnosis of heart failure. Patients who died during the hospital stay or were transferred to other acute care hospitals or hospice care were excluded.P<.001).Readmission occurred in 107  encounters. The out-of-sample area under curve for the 2-stage predictive model was 0.73 (SD 0.08). Dynamic clinical predictors capturing laboratory results and vital signs had the highest predictive value compared with demographic, administrative, medical, and procedural data included. Unsupervised clustering identified four risk trajectory groups: decreasing risk , high risk , moderate risk , and low risk . The decreasing risk group demonstrated change in average probability of readmission from admission (0.69) to discharge (0.30), whereas the high risk (0.75), moderate risk (0.61), and low risk (0.39) groups maintained consistency over the hospital course. A higher level of hemoglobin, larger decrease in potassium and diastolic blood pressure from admission to discharge, and smaller number of past hospitalizations are associated with decreasing readmission risk (Dynamically predicting readmission and quantifying trends over patients’ hospital stay illuminated differing risk trajectory groups. Identifying risk trajectory patterns and distinguishing predictors may shed new light on indicators of readmission and the isolated effects of the index hospitalization. Patients hospitalized with heart failure suffer the highest rates of 30-day readmission among other clinically defined patient populations in the United States . NationaThese past readmission prediction models were static—designed to function at a single point in time—either at the beginning of the patient’s stay at admission where data are limited  or near In this study, we dynamically predicted a 30-day readmission risk on each day of the patient’s hospitalization assuming that the patient was discharged on that day. The reason to predict this counterfactual outcome is that, by definition, 30-day readmission is readmission within 30 days after discharge. Therefore, a discharge time must be defined while predicting 30-day readmission risk. The current day is most appropriate as the discharge day for real-time prediction as care providers are most interested in learning the readmission risk if the patient is to be discharged that day. We can then keep updating this risk daily for the duration of stay. This may be able to aid physicians in their decision on when to discharge a patient.The objective of this study was to develop a predictive model of 30-day readmission for patients with heart failure that functions in real time over the course of a patient’s hospitalization. We made predictions for the risk of 30-day readmission as if the patient were discharged on each day of their hospitalization to illustrate how this risk varied over time  for each patient. We hypothesized that quantifying time-dependent trends in readmission risk had the potential to illuminate the effects of clinical measures and interventions on readmission likelihood at discharge. These predictions could potentially support physician decision making on when to safely discharge patients from the hospital based on the current level of risk and trend over time. These analyses further enabled the identification of groups of patients with heart failure having differing trajectories of readmission risk over their hospital stay.We conducted a retrospective cohort study of patients with a primary diagnosis of heart failure between September 1, 2013, and August 31, 2015, from a community hospital in Maryland. Patients with heart failure were identified using the International Classification of Diseases–ninth revision codes: 428.x, 402.01, 402.11, 402.91, 404.01, 404.03, 404.11, 404.13, 404.91, and 404.93 . PatientAs per the Centers for Medicare and Medicaid Services definition, we defined our primary outcome 30-day readmission as all-cause readmission to the same hospital within 30 days after discharge from the index hospitalization .We derived hypothesized predictors from heart failure and readmission risk factors used in previous studies as summarized in Dynamic predictors comprised variables dependent on the time of prediction and included the elapsed length of stay, vital signs , and laboratory results . Time series characteristics of vital signs and laboratory results were engineered as predictors in the following manner:Number of measurements: n.Average valueStandard deviationMinimum valueMaximum valueNormalized index of the minimum: index of minimal value / nNormalized index of the maximum: index of maximal value / nAverage of last 3 measurementsAverage of first-order differenceNormalized index predictors were designed to capture the relative point during hospitalization when these extreme measurements occurred . For exaAll laboratory tests were not necessarily available for each encounter in the database. For example, 70 out of the 534 encounters did not have proBNP measurement data, 55 did not have AST data, and 37 did not have troponin T data. We excluded laboratory-related predictors with more than 30% unavailable data and imputed missing values with the encounter-based mean for features with less than 30% missing data. Predictors quantifying the number of measurements  were created from the hypothesis that increased measurement intensity might relate to severity of illness and likelihood of readmission . This alOur goal was to build a daily prediction model structured to yield a prediction of 30-day readmission risk each day (6 am) during the patients’ stay based on data available at the time of prediction. Thus, predictions made earlier in the stay are based on less information than predictions made later. We generated predictions at the time of hospital admission, then 6 am each subsequent day, and finally at hospital discharge.The challenge for building a daily prediction model is that the daily outcomes are unknown. Thus, for a particular day, we do not observe whether or not a patient would be readmitted if they were discharged earlier or later than their observed discharge date. To address this uncertainty, we used a 2-stage modeling approach. In the first stage, we estimated the daily risk of readmission based on knowledge of the full encounter data. In the second stage, we used this estimated risk as the outcome for daily prediction using only the data available up to that particular prediction time. We used the first-stage predictions as a proxy for the unobservable outcomes in the second stage.Specifically, in the first stage, we trained a logistic regression model for 30-day readmission based on all of the information available for each encounter at the time of discharge. Backward selection was used to eliminate predictors without predictive value in the first-stage model. Each observation for this training data corresponded to a patient encounter .counterfactual readmission risk as we are estimating this risk assuming that the patients were discharged earlier than their actual observed discharge time. Each observation in the training data for the second-stage model corresponds to a patient day that comprises the cumulative data available up to the prediction time . An example of the second-stage training data is shown in In the subsequent second stage, we trained a beta regression model to predict the daily risk of 30-day readmission based on the risk of readmission predicted (on the same day) by the first-stage model. We call this the P>.05) [Readmission risk from the first-stage model output was bounded between 0 and 1 and followed a beta distribution (Kolmogorov-Smirnov goodness-of-fit test P>.05) . Thus, bBoth the first-stage and second-stage models were developed using 5-fold cross-validation . Predictive performance measures of the AUC for the first-stage model (encounter-level), and pseudo R-squared value for the second-stage model (daily) were evaluated out-of-sample. R-squared represents the squared correlation coefficient between the counterfactual daily readmission risk and predicted values from the beta regression model.A patient’s daily 30-day readmission risk may change over the course of their hospitalization because of the evolution of their condition as therapies are administered. A total of 78.4%  of all laboratory tests, 74.0% (2173/2935) of all procedures, and 92.5% (3937/4258) of all medication orders occurred before the end of day 5 for our study cohort. Thus, we believe that the majority of variation in readmission risk occurred before this time. After training the beta regression model in the second stage, we used the trained model for predicting the daily readmission risk at admission—day 1 to day 5—and discharge. We applied unsupervised clustering using the K-means algorithm to determine readmission risk trajectories over these 7 specific milestones during a patients’ stay . We usedDetailed steps of the entire modeling process are shown in We conducted all of our analysis using Python (version 2.7) and R (version 3.4.1).In our sample, we observed 534 patient encounters by 454 unique patients that included 164 patients who experienced multiple encounters. In total, 30-day readmission occurred in 107  of these encounters. Characteristics of the patient cohort stratified by readmission outcome may be seen in Logistic regression with backward selection identified an optimal set of 57 predictors for our first-stage encounter-level prediction model. These optimal predictors are displayed in decreasing risk group with 131 encounters  was unique in demonstrating appreciable change in risk over the hospitalization from admission (.69 probability of readmission) to discharge (.30). The remaining trajectory groups maintained a consistent readmission risk over time. This included the high risk group of 113  encounters with average readmission risk maintained above 0.75 over their course of care. The low risk cluster with 113  encounters were admitted with a relatively low average readmission risk of 0.39 that decreased to 0.21 at discharge. The moderate risk cluster had 177  encounters, and its average readmission risk had a mean of 0.61.A total of 4 distinct trajectories emerged from the unsupervised clustering analysis as seen in P<.001) as seen in Discriminant analyses demonstrated 18 predictors with similar characteristics across the 4 risk trajectory groups  for our dynamic modeling approach was 0.73 SD 0.08), which was relatively high but within the range  recognizing high readmission risks associated with patients who have been hospitalized frequently in the past, (2) abnormally low hemoglobin that is either consistent throughout the stay or drops near discharge is associated with higher risk of readmission, (3) abnormally low sodium is associated with high readmission risk, (4) abnormally high diastolic blood pressure late in the hospital stay or no considerable decreases (<4 mmHg) from admission to discharge may indicate higher readmission risk, (5) abnormally high potassium levels near discharge may indicate high risk of readmission, and (6) a minimal potassium occurring closer to discharge is associated with decreasing risk of readmission.There were several limitations in our study data and methods that should be considered when interpreting results. First, clustering of time-dependent trends in readmission used up to 7 time points  for each encounter. This approach was advantageous in enabling comparisons of readmission risk profiles across patients but limited in its ability to compare short-stay patients  with longer-stay patients (>5 days). Ideally, we would perform clustering analysis controlling for the entire duration of each patient’s stay. However, we believe that our 7–time point approach yielded results that would not qualitatively change as readmission risk trends vary less toward the end of the hospitalization, particularly for longer-stay patients. Second, owing to the limited sample size, we treated each encounter in our first-stage model as independent. When the same patient has multiple encounters, as seen in our data, this assumption may be contravened. This becomes particularly important to avoid bias in out-of-sample performance evaluation . However, we determined that treating each patient encounter independently was analogous to how this predictive tool could be run in real time. We also further mitigated risks of bias by performing 5-fold cross-validation. Third, we used mean imputation to impute missing data in features. The disadvantage of mean imputation is that it reduces the variance of the imputed features and, as a result, the discriminability of that feature. A more comprehensive method to impute missing data may improve the prediction accuracy. Finally, evaluating predictive performance of our second-stage predictive model presented challenges methodologically. We were unable to observe the counterfactual readmission outcomes . Using the last day’s outcomes to estimate the predictive performance of the second-stage model is an incomplete estimate, as we ignore the predictive performance on all days before the day of discharge. Therefore, we used a pseudo R-squared value to estimate the predictive performance of the second-stage model.We applied supervised machine learning using a novel 2-stage approach to construct a 30-day readmission prediction model that yields daily predictions for patients with heart failure over time. We estimated the unknown counterfactual daily readmission risk using an encounter-level logistic regression model (first stage). A beta regression model using predictors engineered from EHR data accumulated to the prediction time  was fit to form the daily readmission risk prediction model (second stage). Investigating dynamic trends in readmission risk over time via unsupervised clustering, we uncovered for distinct groups based on readmission risk trajectory: decreasing, high, moderate, and low. The clinical features distinguishing these groups  may shed light on new indicators of readmission for patients with heart failure and the isolated effects of the index hospitalization. Our ultimate goal is to use the important features and our model to develop a clinical tool for dynamically predicting readmission for patients with heart failure and target patients with high readmission risk. The clinical tool is aimed to show readmission risk for each patient and also the corresponding factors that are associated with high or low readmission risk. This study provides a first step in the development of such a tool by proposing a dynamic prediction model for readmission risk of patients with heart failure.


Can adjustment of circadian timing through light flash therapy during sleep increase total sleep time in teenagers?In this double-blind, placebo-controlled, randomized clinical trial of 102 adolescents, light flash therapy alone was ineffective. When delivered in combination with a cognitive behavioral therapy meant to encourage an earlier bedtime, the combination of light flash therapy and cognitive behavioral therapy significantly and stably increased total sleep time by nearly 45 minutes per night.Combination light flash therapy and cognitive behavioral therapy is an effective tool to increase sleep time and combat sleep loss in teenagers. Owing to biological, behavioral, and societal factors, sleep duration in teenagers is often severely truncated, leading to pervasive sleep deprivation.To determine whether a novel intervention, using both light exposure during sleep and cognitive behavioral therapy (CBT), would increase total sleep time in teenagers by enabling them to go to sleep earlier than usual.This double-blind, placebo-controlled, randomized clinical trial, conducted between November 1, 2013, and May 31, 2016, among 102 adolescents enrolled full-time in grades 9 to 12, who expressed difficulty going to bed earlier and waking up early enough, was composed of 2 phases. In phase 1, participants were assigned to receive either 3 weeks of light or sham therapy and were asked to try to go to sleep earlier. In phase 2, participants received 4 brief CBT sessions in addition to a modified light or sham therapy. All analyses were performed on an intent-to-treat basis.Light therapy consisted of receiving a 3-millisecond light flash every 20 seconds during the final 3 hours of sleep (phase 1) or final 2 hours of sleep (phase 2). Sham therapy used an identical device, but delivered 1 minute of light pulses  per hour during the final 3 hours of sleep (phase 1) or 2 hours of sleep (phase 2). Light therapy occurred every night during the 4-week intervention. Cognitive behavioral therapy consisted of four 50-minute in-person sessions once per week.Primary outcome measures included diary-based sleep times, momentary ratings of evening sleepiness, and subjective measures of sleepiness and sleep quality.Among the 102 participants , 72 were enrolled in phase 1 and 30 were enrolled in phase 2. Mixed-effects models revealed that light therapy alone was inadequate in changing the timing of sleep. However, compared with sham therapy plus CBT alone, light therapy plus CBT significantly moved sleep onset a mean (SD) of 50.1 (27.5) minutes earlier and increased nightly total sleep time by a mean (SD) of 43.3 (35.0) minutes. Light therapy plus CBT also resulted in a 7-fold greater increase in bedtime compliance than that observed among participants receiving sham plus CBT , as well as a mean 0.55-point increase in subjective evening sleepiness as compared with a mean 0.48-point decrease in participants receiving sham plus CBT as measured on a 7-point sleepiness scale.This study found that light exposure during sleep, in combination with a brief, motivation-focused CBT intervention, was able to consistently move bedtimes earlier and increase total sleep time in teenagers. This type of passive light intervention in teenagers may lead to novel therapeutic applications.NCT01406691ClinicalTrials.gov identifier:  This randomized clinical trial examines whether a novel intervention, using both light exposure during sleep and cognitive behavioral therapy, increases total sleep time in teenagers by enabling them to go to sleep earlier than usual. Associations have been established between sleep duration and teenagers’ grade point average,5 physical health,7 substance use and risk-taking behaviors,9 and psychological well-being.12Insufficient sleep in adolescence is increasingly recognized as a public health concern, with implications for academic performance, health, safety, and psychological well-being. Across the previous 2 decades, sleep durations in adolescents have been declining.14 even as the need for sleep remains constant or increases.15 This decrement in sleep is likely owing to a combination of later bedtimes, coupled with fixed, earlier school start times,16 which results in insufficient time for sleep. Later bedtimes are thought to be caused by a set of reinforcing influences: a naturally occurring delay in the timing of the circadian clock16 and increasing psychosocial activities at night .17 These nocturnal activities can laten bedtime both directly and indirectly, owing to the concomitant evening light exposure that further delays the timing of the circadian clock.Multiple factors contribute to insufficient sleep in adolescence. Empirical data indicate that adolescents sleep less with age,19 or through administration of in-class morning light therapy,20 have not been successful in changing weekday sleep durations. One trial using light therapy and cognitive behavioral therapy (CBT) improved the sleep of adolescents meeting criteria for delayed sleep phase disorder,21 but its applicability to a broader population of teenagers with sleep difficulties is unknown. Although the use of CBT for insomnia is a widely validated method,22 it is unlikely to be useful as a stand-alone treatment for delayed sleep, which is not equivalent to insomnia, as there are innate biological reasons  that would be difficult to overcome with behavioral efforts. Use of behavioral therapy to promote sleep at an earlier circadian phase (without shifting the clock to an earlier phase) may, in fact, lead to the induction of insomnia.23Previous interventions have attempted to address this latening of bedtime using a combination of early morning phototherapy (to move the timing of the circadian clock to an earlier clock hour) and behavioral or education-based interventions , with mixed results. Classroom-based interventions, designed to improve sleep through education25 In theory, this exposure would realign bedtime to a time more amenable to getting sufficient sleep. Such a change in circadian timing, however, would be temporary and would need to occur each day to offset the naturally occurring biological delay associated with adolescence. Also, traditional light therapy often requires teenagers to awaken earlier than their habitual wake time, which engenders even greater sleep loss. However, one study has shown that the human circadian system can respond to sequences of extraordinarily brief, millisecond-length flashes of light while people are sleeping (flash therapy), and that such light does not disrupt sleep.26 The physiological circuit underlying the circadian responses to light flashes is not completely understood, but the ability of the neurons that project from the retina to the suprachiasmatic nucleus to continue their electrical response for several minutes after cessation of light27 is likely to be in part responsible for the integration of light flashes over time . Using flashes of light is a powerful tool with which one can manipulate the circadian system, as it is more than 2- to 3-fold stronger than continuous light.28 Thus, flash therapy delivered during the last hours of habitual sleep could set the human circadian pacemaker to an earlier hour, allowing teenagers to go to sleep earlier, and without the burden of having to wake earlier than usual.Traditional bright light phototherapy in the morning is designed to offset the natural delay in circadian timing (and bedtime) by evoking an advance in the timing of the circadian clock . In adolescents, exposure to bright light prior to their habitual wake time, and to a lesser degree after awakening, will advance the timing of the circadian clock.Our study consisted of 2 phases. Phase 1 examined whether, independent of any behavioral adjustments, flash therapy during sleep could move sleep timing earlier and increase total sleep in teenagers. Phase 2 examined whether flash therapy in combination with brief CBT could move sleep timing earlier and increase total sleep time in teenagers. In both phases, participants had 1 week of ad libitum baseline sleep monitoring at home with a written diary followed by 4 weeks of treatment. The primary outcome measures were the timing of the onset of sleep and the total amount of sleep time.29 Assent was obtained from the adolescent participants and consent was obtained from at least 1 parent of each participant. This study followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline.Phase 1 included 72 adolescents aged 14 to 18 years , while phase 2 included 30 adolescents aged 14 to 18 years   trial p. RecruitStudy procedures are illustrated in Participants visited the laboratory with a parent for a baseline evaluation, followed by 1 week of ad libitum baseline sleep monitoring at home with a written sleep diary. At the end of this week, a technician visited the participant’s home to install the light device in the participant’s bedroom. Once the light was placed, participants were instructed to go to bed 1 hour earlier than their usual bedtime for the remainder of the study, to allow opportunity for the light to take effect. In phase 1, participants were called weekly to fill out questionnaires on sleepiness and sleep quality, and to report whether they noticed the light and whether the light interfered with their sleep. In phase 2, instead of weekly telephone calls, participants came to the laboratory for weekly therapy sessions  during which the same questionnaires were completed. At the end of the 4-week intervention, participants returned to the laboratory with a parent to complete an end-of-treatment evaluation. Participants completed all portions of the study during the academic year and not during scheduled winter and spring breaks. If a teenager slept in 2 households, the light was placed in each household at approximately the same position relative to the bed.30 Adolescent Sleep-Wake Scale ,31 Cleveland Adolescent Sleepiness Questionnaire (daytime sleepiness),32 Credibility/Expectancy Questionnaire (participant belief in and expectation of results from intervention),33 and a single question inquiring about the reasons for sleep difficulty. During the course of the study, participants completed a sleep diary34 every morning, answered a question about sleepiness delivered via text message 2 hours prior to baseline bedtime every evening ,35 and repeated the Adolescent Sleep-Wake Scale and Cleveland Adolescent Sleepiness Questionnaire each week of the intervention and again at the end of treatment. The Credibility/Expectancy Questionnaire was also repeated at the end of treatment. At baseline and the end of treatment, a parent completed the Child and Adolescent Sleep Checklist for parents (parent’s impression of participant’s sleep).36At a baseline visit, participants completed the following questionnaires: Morningness-Eveningness Scale for Children (morning vs evening preference),28 The light timers for both groups were preprogrammed by one of us (J.M.Z.) who was not involved in assessment at any point. The placement of the experimental light was done to minimize the distance between the light and normal sleeping position and was measured for each participant.Participants were randomized to receive an active or sham light stimulus for the duration of the treatment. In the active condition (light), daily light therapy  in the form of brief light pulses began 3 hours (in phase 1) or 2 hours (in phase 2) before the participant’s target weekday wake time. Approximately 4000 lux of broad-spectrum white light (approximately 200-600 lux at the cornea after eyelid filtration) was delivered in each 3-millisecond flash, occurring 20 seconds apart. Light was generated by a custom-designed xenon flash bulb . The sham group received 1 minute of light pulses  each hour; this dose is believed to be insufficient to shift the circadian system.Consistent with other brief behavioral treatments for sleep disturbances, the CBT intervention consisted of four 50-minute, in-person sessions once per week delivered by a licensed clinical psychologist or trained doctoral student . The CBT intervention had several core components, including (1) education on the circadian system, the effect of light, and physiological processes; (2) information about the role of sleep in domains relevant to adolescents ; (3) sleep hygiene and stimulus control, well-established insomnia treatment components designed to improve sleep and associated contextual cues; and (4) activity scheduling to wake up earlier on weekends and anticipate obstacles.37 teenagers and parents, together with the study therapist, set collaborative goals for the teenagers’ sleep at the baseline evaluation. To further promote change, teenagers were asked at the baseline evaluation to reflect the importance of sleep and their level of confidence and readiness to make sleep changes. Motivational interviewing was woven into each subsequent treatment session, including exploring ambivalence about change where resistance was met. At baseline, teenagers completed a worksheet on personal strivings,38 listing domains that were important to them . The treatment was subsequently tailored to adolescents’ individual goals, and the therapist could highlight relationships between adequate sleep and athletic performance, mood, or academic pursuits, as dictated by the adolescent.Motivational interviewing and values-based clarification were central features of the CBT intervention. Given that motivational interviewing to promote behavior change may be strengthened by parent participation,t tests for continuous data and χ2 tests for categorical data. Differences in baseline measures were also evaluated by calculating the standardized mean difference (SMD); an SMD of 0.2 is considered small.39 All analyses were based on the intent-to-treat model. For investigating changes in sleep diary variables, Ecological Momentary Analysis, and weekly sleep questionnaires, linear mixed-effects models allowing for random intercepts were used. The differential effect of treatment was also expressed as Cohen d, calculated by dividing model-estimated difference in mean change by observed pooled SD at baseline.40 Mixed-effects models were run in R, version 3.1.2 , using lme4, version 1.1-7. Two-tailed P values were generated using Satterthwaite’s approximations41 for degrees of freedom in lmerTest, version 2.0-29. P < .05 was considered significant.Group differences in demographic variables were analyzed using To examine variability across the intervention period, we calculated for each individual the proportion of days in which adolescents were compliant with prescribed bedtimes, defined by attempts to sleep at or before the target bedtime. As target bedtimes and wake times were collaboratively set in treatment sessions in phase 2, working toward an advance of 60 minutes, we further calculated a ratio of bedtime compliance relative to wake time compliance. For example, an individual who woke at the target wake time on 20 days and went to sleep at the target bedtime on 10 days would have a ratio of 0.5. This ratio would reflect compliance with sleep initiation (thought to be under circadian and self-motivated control) relative to compliance with sleep termination (more fixed through school start time). Ratios were log-transformed prior to statistical analyses.P = .52), subjective sleep quality , and parents’ impression of their teenager’s sleep . There was no singular reason teenagers gave for being unable to go to sleep earlier than their current bedtime. One teenager in phase 2, randomized to the sham plus CBT group, was excluded from analyses after reporting that an illness during both the baseline and posttreatment periods yielded unrepresentative sleep.Among the 102 participants , 72 were enrolled in phase 1 and 30 were enrolled in phase 2. Study flow is depicted in P < .001), which was different from the 1% change in the sham group , although groups differed on this variable by 3% at baseline   . In phast25 = −3.13; P = .006). This finding was also reflected in significantly greater variability in bedtimes in the sham plus CBT group (11.5 minutes) compared with the light plus CBT group . Wake time variability did not differ between the groups .In phase 2, compared with the sham plus CBT group, the light plus CBT group showed greater improvement in sleep. The light plus CBT group had significantly earlier sleep onsets , greater total sleep time , and earlier attempts to initiate sleep  . As an eP = .01) (P = .02) with increasing sleepiness in the light plus CBT group  and a nonsignificant decrease in sleepiness in the sham plus CBT group  during the evening  evening sleepiness ratings  . In phas evening .P = .007) but not the sham plus CBT group  (All groups from both phases reported reductions in daytime sleepiness (Cleveland Adolescent Sleepiness Questionnaire) and increases in sleep quality  . There wr = 0.11; P = .53) or phase 2 . In phase 1, more individuals in the light group relative to the sham group reported either noticing the light  or having their sleep disturbed by the light . To account for this disruption, the duration of the light exposure was reduced in phase 2. Subsequently, we found no differences in the number of teenagers in the light plus CBT group and in the sham plus CBT group who reported either seeing the light  or having their sleep disturbed by the light , although the low number of participants preclude us from concluding that there was no effect of the light on sleep.Light placement in participants’ bedrooms was a mean (SD) distance of 91.3 (62.2) cm (phase 1) and 111 (71.2) cm (phase 2) from participants’ pillows. There was no correlation between light placement and change in sleep onset time in either phase 1  or at the end of segment 4 .In phase 2, the intervention was rated favorably, with both groups demonstrating high mean (SD) Credibility/Expectancy Questionnaire scores at the end of segment 1  and at the end of segment 4 . There were no differences in Credibility/Expectancy Questionnaire scores between the groups at the end of segment 1  and phase 2 (2 hours) are not identical, it would be important to examine whether 2 hours of light flashes were sufficient to change the time of sleep onset without the administration of adjunctive CBT. The calendar dates of therapy administration were not identical between phase 1 and phase 2, precluding direct comparison.In phase 1, light flashes were both ineffective and somewhat disruptive to sleep. In response to this shortcoming, we decreased the duration of the flash sequences from 3 hours to 2 hours, which appeared to alleviate the negative effects of the light on sleep. Not only was there no greater notice or disruption of the light flashes, but teenagers also slept to the same clock hour in both conditions, indicating that the light flashes did not specifically disrupt the end of sleep (when the flashes were being delivered). It is also possible that shortening the flash sequence increased its effectiveness by reducing the likelihood that the flashes had either no effect or had a phase-delaying effect on circadian timing .42 We have found more recently that a flash sequence occurring every 8 seconds is able to produce a change in circadian timing that is twice as large as the flash sequence used in the current study. If needed, bedtimes could be moved even earlier and more sleep could be obtained using an optimized light flash sequence, although convincing teenagers of the need for a further truncation of evening wake time could be difficult.Future studies should include objective measurements of sleep , given the possibility of reporting bias, as well as long-term monitoring of the light intervention. This latter improvement would be an important next step to determine ongoing acceptance of the light therapy and to determine whether the beneficial effects of light plus CBT could be maintained for months or years. The light sequence that we used (one 3-millisecond flash every 20 seconds for 2-3 hours) was selected based on early data.The light flashes in this study were delivered through a customized beacon. Although we did not observe any associations between the placement of the beacon in the adolescents’ rooms and any of our measures, it is possible that this is not an ideal form because the beacon is not commercially available and could affect other individuals sleeping in the same room  who might not want to be affected by the light. Development of an alternative light delivery system  might be necessary in such conditions.47 There are shortcomings to each of these recording modalities, and the use of a combination of them may be necessary, especially in examination of the daytime sequelae of sleep deficits , which is also an important area of follow-up.As previously mentioned, the use of objective sleep monitoring in a larger number of individuals will be critical in future studies of the effect of this intervention. Subjective sleep monitoring, actigraphic sleep monitoring, and polysomnographic sleep monitoring have some overlapping sources of variance but can be independent of one another, especially under conditions of poor sleep.Despite the potential limitations of the light delivery mechanism, when combined with CBT, light flashes at the end of sleep enabled an earlier bedtime and increased nightly sleep by approximately 45 minutes in teenagers. This type of passive light intervention in teenagers may lead to novel therapeutic applications.