Roofers are at increased risk for various malignancies and their occupational exposures to polycyclic aromatic hydrocarbons (PAHs) have been considered as important risk factors. The overall goal of this project was to investigate the usefulness of phosphorylated histone H2AX (γH2AX) as a short-term biomarker of DNA damage among roofers.Blood, urine, and dermal wipe samples were collected from 20 roofers who work with hot asphalt before and after 6 h of work on Monday and Thursday of the same week (4 sampling periods). Particle-bound and gas-phase PAHs were collected using personal monitors during work hours. γH2AX was quantified in peripheral lymphocytes using flow cytometry and 8-hydroxy-2-deoxyguanosine (8-OHdG) was assessed in urine using ELISA. General linear mixed models were used to evaluate associations between DNA damage and possible predictors . Differences in mean biomarker and DNA damage levels were tested via ANOVA contrasts.Exposure measurements did not show an association with any of the urinary biomarkers or the measures of DNA damage. Naphthalene was the most abundant PAH in gas-phase, while benzo(e)pyrene was the most abundant particle-bound PAH. Post-shift levels of γH2AX and 8-OHdG were higher on both study days, when compared to pre-shift levels. Cigarette smoking was a predictor of γH2AX and urinary creatinine was a predictor of urinary 8-OHdG. Between-subject variance to total variance ratio was 35.3 % for γH2ax and 4.8 % for 8-OHdG.γH2AX is a promising biomarker of DNA damage in occupational epidemiology studies. It has a lower within-subject variation than urinary 8-OHdG and can easily be detected in large scale groups. Future studies that explore the kinetics of H2AX phosphorylation in relation to chemical exposures may reveal the transient and persistent nature of this sensitive biomarker of early DNA damage.The online version of this article (doi:10.1186/s12940-016-0182-4) contains supplementary material, which is available to authorized users. Workers around the world experience daily exposures to potentially carcinogenic chemicals. Identifying the role of these exposures in cancer development later in life has been a major challenge in occupational epidemiology. Estimating the exposure-cancer association becomes more complicated by simultaneous exposures to other environmental and lifestyle factors. A further challenge is the long latency period between carcinogenic exposure and cancer diagnosis. Many times occupational studies rely on estimates of current exposures and their association to short-term markers of health effects. Among these markers, measures of DNA damage are viewed as reliable indicators of increased cancer risk , as theyRoofers are at increased risk for different malignancies such as lung, bladder, stomach, skin and buccal cavity cancers, and leukemia –10. ExpoExposure to PAHs can increase reactive oxygen species (ROS) formation in the body. When the cellular antioxidant defense system is disturbed, the increased amount of ROS can cause oxidative damage to biomolecules such as DNA, proteins and lipids. Recent studies have supported the link between insufficient cellular defense towards oxidative DNA damage and increased susceptibility to cancer development –33. UrinDouble-strand DNA breaks have also been linked to PAH exposures; this form of DNA damage can be measured using phosphorylated histone H2AX (γH2AX) in individual cells . IncreasBefore a biomarker can be comfortably used in epidemiology studies, it needs to be validated based on the following criteria: 1) The relationship between the biomarker and exposure in question, 2) The formation, distribution and elimination of the biomarker in humans, 3) Variation of the biomarker between- and within- study participants, 4) Baseline values of the biomarker in the general population, and finally 5) Cost and difficulty of analytical techniques . Here, wTwenty roofers employed by one roofing company were recruited. The study site was a roof replacement project located in Colorado Springs, Colorado and was visited by the field study team over four weeks between July and September of 2013. Potential participants were informed about the study at the site and those who signed the informed consent under University of Colorado’s IRB (COMIRB) approved protocol (COMIRB Protocol # 12–0443) were recruited. Each week, a new group of workers participated in the study over two workdays: Monday and Thursday. Study questionnaires were administered before and after the work-shift in either English or Spanish; the latter applied by a Spanish speaking interviewer. Biological samples (urine and blood) were collected before and after 6 h of work. Study participants also provided hand wipes (with 3 ml sun flower oil) at each sampling period. Information on personal characteristics , life-style factors , use of protective equipment during the study day  and specific work tasks performed during the day  were collected via questionnaires. The before-work questionnaire focused on non-occupational sources of PAH exposures and the after-work questionnaires contained more detailed questions on work practices. Both of these questionnaires are provided in Additional file After the completion of morning questionnaires and collection of biological samples, the participants were given lightweight vests with air monitors to collect air samples from within each worker’s breathing zone air during the shift. The participants were then asked to return to their work. After 6 h of work participants returned the vests with the air monitors. The total number of samples collected from 20 participants is as follows: 79 urine, 79 blood, 40 air, and 79 dermal wipes. One of the participants had to leave the site on the second study day due to a family emergency and could not provide samples that afternoon (period 4).2.5 sampling inlets  and 37 mm Teflon filters. Gas-phase PAHs were collected immediately downstream of the filters using standard adsorbent tubes . Method details are provided in Additional file Polycyclic aromatic hydrocarbons (PAHs) in ambient air were measured within the breathing zone of workers via personal sampling. Particle-bound PAHs, 4-ring and above, were collected using personal sampling pumps (SKC XR5000) fitted with PMPAH metabolites were analyzed using an automated solid-phase extraction based on a method developed by Romanoff . DetailsDermal exposure samples were collected using a previously published hand washing method with sunflower oil . Detailsn = 80) were tested to evaluate levels of γH2AX. This method was optimized by treating fresh lymphocytes, in triplicate from one volunteer who was not a roofer, with various amounts of H2O2 (0.02-0.24 mM) and freezing via the same method described above. Frozen samples were thawed in a 37 °C water bath  and 500,000 cells were added to wells in a round-bottom 96 well plate . Freezing media was removed and cells were washed 3 times with PBS. All washes and buffer removals involved a 600 g spin for five minutes at room temperature. Cells were then fixed with 200 μL BD Cytofix fixation buffer (BD Biosciences) and incubated for 15 min at room temperature. Next, the fixative was removed, and cells were washed twice with 200 μL of PBS. Cells were then permeabilized with 200 μl −20 °C Perm Buffer III (BD Biosciences) for 5 min at RT. After one wash in 200 μL of 1x perm/wash buffer (BD Biosciences), 200 μl of 1x stain buffer (BD Biosciences) was added to each well to block non-specific binding. After 20 min at room temperature the cells were washed two times with 200 μl of Perm/Wash buffer. Next, 100 μl stain buffer and 5 μl BD antibody  were added and cells were incubated for 60 min at room temperature in the dark. After antibody removal, wells were washed three times with 200 μl of 1x perm/wash buffer. Finally, the cells were resuspended in 300 μl FACS fix  and read with CFlow Plus software on a C6 flow cytometer . All samples were run in triplicate and results are given in mean fluorescence intensity (MFI) of the lymphocyte gated FL1 channel . Plots for publication were made using the FCS Express4 Flow Research Edition software. Figure Methodological details on processing of peripheral blood samples are described in Additional file All statistical analyses were conducted using SAS system software  at a significance level of 0.05. All tests were performed after  logarithmic transformation of urinary analytes , γH2AX and PAH levels  to satisfy the normality assumption, and data were summarized as geometric means (GMs) and geometric standard deviations (GSDs). Average levels of naphthalene and pyrene in personal breathing zone samples or on dermal wipes were compared between the two study days or between smokers and nonsmokers for each day, using Student’s t-tests. Specific contrasts  were applied to test for differences in log-transformed mean levels of urinary biomarkers and DNA damage measures by sampling period (before and after the work-shift on first and second day) and cigarette smoking status. For this purpose, a two-way analysis of variance (ANOVA) procedure was applied for each day separately. For each sampling day, Pearson’s correlation coefficients (with 95 % CI) were used to measure the strength of association between different pairs of exposure and biomarker measurements.In personal air samples using filters (FLT) 42.5 % of naphthalene measurements and 35 % of benzo(e)pyrene measurements were below limit of detection (LOD). For personal air XAD samples 42.5 % of pyrene was below LOD, while naphthalene was detected in all of the XAD samples. For dermal wipes, 3.5 % for naphthalene samplers were below LOD and pyrene was detected in all of the samples. Measurements of γH2AX (lymphocytes) and 8-OHdG (urine) were above detection limit for all of the samples. For urinary biomarkers: 1-OHPyr was below LOD in 24 % of the samples while 1-OHNap was below LOD in one urine sample (1.2 %). 2-OHNap and creatinine were both detected in all of the urine samples. For samples that were below the LOD, a proxy measurement was assigned using the value of LOD/√2 before statistical analyses .Information for a number of general and work-related variables was collected via questionnaires  were retained. Backward selection of all retained independent variables and their plausible two-way interactions were used to achieve final models (using a significance level of p < 0.05). Multivariable models had the general form:Repeated-measures general linear mixed modeling (PROC MIXED) was used to examine associations between DNA damage measures (γH2AX and 8-OHdG) and sampling period (before/after work on two separate sampling days) adjusting for confounders. For the general linear mixed models only confounders with values at the four different sampling periods were considered. Candidate variables that were considered amounted to eight variables for the model of γH2AX , and nine variables for the model of urinary 8-OHdG , plus the two-way interactions between urinary biomarkers and cigarette smoking in both models. The most likely candidate variables were screened as follows. First, DNA damage measures were regressed on each covariate separately and variables that suggested significant contributions (Yi represents the subject-specific mean of log-transformed levels of γH2AX (or 8-OHdG) for the ith subject at sampling period jh, α is the intercept representing the average level of Yi when all independent variables are zero for an average worker, bi is the random intercept for subject i that captures the heterogeneity between individuals, βk is the regression coefficient for the kth independent variable Xik for the ith subject, and εij is the error term. Given the sample size of 80 measurements from 20 individuals, a linear mixed model with about 4–6 predictors will likely be a stable model. Estimates of the percentage of variance explained by each of the significant covariates in the linear mixed models were calculated using the conditional and marginal formulas of R2 [where as of R2 . The intp-value < 0.0001 for difference). When divided by smoking status, post-shift levels of 8-OHdG remained to be higher than those observed pre-shift in nonsmokers  and in smokers , difference was statistically significant for both groups (p < 0.05). The overall difference between post-shift and pre-shift levels was smaller for γH2AX  and remained small in nonsmokers  and in smokers .Levels of DNA damage markers were at higher concentrations in samples collected after work when compared to those observed before work. The overall difference between post-shift and pre-shift levels was 1.7-fold for urinary 8-OHdG , naphthalene was the most abundant PAH, quantified in all of the samples. Pyrene on the other hand, was only quantified in 57.5 % of the XAD samples. For PAH measurements obtained from filters (representing particulate phase) the most abundant compound was benzo(e)pyrene quantified in 65 % of all samples, followed by naphthalene quantified in 57.5 %. Only 35 % of filter samples had quantifiable levels of pyrene. Table Levels of naphthalene on dermal wipes did not differ significantly before or after the work on both study days Table . Levels Table p = 0.005, Additional file All urinary biomarkers were higher after work on Monday in both smokers and nonsmokers, but the post-shift to pre-shift difference was statistically significant only for urinary 1-OHPyr and 8-OHdG in smokers .For γH2AX, according to the conditional RIn models of urinary 8-OHdG measurements, cigarette smoking was not a significant predictor, but urine creatinine had a great impact on the levels and was kept in final models. Sampling period also remained a significant predictor. When compared to Monday morning, levels collected on Monday afternoon corresponded to 55 % higher levels of urinary 8-OHdG, and levels collected at the end of the week (period 4) were increased by 50 %. The percentage of the between-workers variation of 8-OHdG in the log scale was 4.8 % of the total variance Table .Based on our model .For the analysis of logged 8-OHdG, according to the conditional RWe have recently shown that urinary 8-OHdG is a promising biomarker reflecting early effects of occupational exposures to PAHs during a single work day . Here weIn this study, sampling period, reflecting four different time points within one workweek, was an important predictor of both DNA damage markers. Other important predictors were cigarette smoking for γH2AX and urinary creatinine for urinary 8-OHdG. Our results once again support that urinary 8-OHdG is highly affected by urine dilution. This is an important concern since many times roofers are exposed to heat and can be dehydrated during the course of a single work day.We also observed that about 35.3 % of the unexplained variance of γH2AX was between subjects, while this number was only 4.8 % for urinary 8-OHdG. The proportion of within-subject variance appears larger for urinary 8-OHdG. These two measures cannot be directly compared as they reflect different types of DNA damage  and are measured in different biological media (γH2AX from lymphocytes and 8-OHdG in urine). However, a low ICC value reflects high within-individual variation of a biomarker and is a sign of poor reproducibility . Other sLevels of pyrene on dermal wipes were higher after work when compared to before work measures. This increase was not observed for naphthalene. Consistently, urinary 1-OHPyr levels significantly increased over work hours on Monday, while the increase in urinary naphthalene metabolites was small. Naphthalene is the most abundant PAH in many environments and naphthalene based biomarkers can potentially increase sensitivity of assays. However, our results in this population suggest that environmental influences and cigarette smoking can overwhelm those of occupational exposures to naphthalene. Results of this study are consistent with our previous findings that urinary 1-OHPyr is a promising biomarker of occupational exposures in roofers and that dermal contact can be an important exposure route .Urinary metabolites of PAHs, particularly urinary 1-OHPyr, are established biomarkers of occupational exposures. Overall, levels of PAH exposures in this study were lower than previous reports in asphalt exposed workers , 72. WhiDespite the increase in urinary biomarkers during work hours, we did not observe consistent correlations between measures of exposure, urinary metabolites, and DNA damage, making it difficult to reach a final conclusion on the association between exposure and biomarkers. However, this is possibly related to the overall low occupational exposures observed during the study period. Consistently, Monday morning levels of urinary 8-OHdG were are approximately 36-fold lower than those observed in our previous study in roofers .Cigarette smoking is an important factor to consider when analyzing γH2AX. The correlation between γH2AX and urinary metabolites of naphthalene on the second study day was possibly due to their common association with cigarette smoking. This is supported by the fact that levels of urinary 1- and 2-OHNap among smokers were much higher before the work shift on Thursday when compared to levels after the work. It is tempting to limit future evaluations to nonsmokers. However, considering the high proportion of smokers  and the There are some limitations of this study that constrain the interpretation of results. The most important limitations are the low levels of exposure observed during the study period and the small number of participants. Repeated sampling at four different time points, however, provided us with a larger effective sample size. We also used ANOVA to test for differences between measurements conducted before and after the work shift without adjustment for repeated measures. While before and after-work samples do not reflect identical conditions, they are also not independent. However, this was only a preliminary approach that has been addressed by the use of linear mixed models where the specific contrasts of interest have been tested. We also recognize that we are conducting some hypothesis tests without adjustment for multiple comparisons, which in turn might produce a few false positive associations.Another limitation is the widespread environmental exposure to PAHs and other toxicants that cause DNA damage. 1-OHPyr has been widely viewed as the gold standard biomarker of PAH exposures –77. NaphThe PAH biomarkers studied here reflect short-term exposures. The estimated elimination half-lives for urinary 1- & 2-OHNap and 1-OHPyr are around 4 h  and 13 hWhile γH2AX is an early response to genotoxic insults, a number of non-occupational factors can contribute to the DNA double strand breaks (DSBs), such as ultraviolet light (UV), environmental chemicals, and even endogenous triggers of DNA damage. Tobacco smoke, a common source of PAHs, is also a potent inducer of DSBs. With the high number of factors influencing γH2AX response, it is important to distinguish between baseline levels in the general population and γH2AX kinetics following specific exposures. Two types of γH2AX foci have been reported previously: the fast transient γH2AX foci associated with rapid repair which takes place within minutes or hours, and the residual foci that persist for several days or months . The majOur overall goal was to explore the usefulness of γH2AX as a possible marker of DNA damage in workers exposed to PAHs using a high throughput flow cytometry assay. One of our evaluation criteria was its association with exposure data, which we could not observe for γH2AX. Urinary 8-OHdG, a commonly used marker of DNA damage, was also not associated with exposure levels in this group. It is possible that the relatively low levels of exposures may have impacted our analyses. As a second criterion, we evaluated the between- and within-subject variation of γH2AX. Here we observed that γH2AX has a smaller within-worker variation when compared to urinary 8-OHdG. Our analyses also confirmed that baseline values of γH2AX are easily detectable in this population, using an inexpensive method such as flow cytometry. Despite the lack of association with exposure data, we propose that γH2AX is a sensitive biomarker of early DNA damage related to occupational exposures. The low within-subject variation, easy and high throughput methodology makes γH2AX a feasible alternative in epidemiology studies. We perceive the need for additional studies to understand baseline values of γH2AX, between- and within-individual variation in different study populations, and the impact of developmental and degenerative diseases, as well as dietary, environmental, and life-style factors on this promising biomarker . This wi


This paper focuses on nano-morphology-controlled small-molecule organic solar cells without solvent treatment for high power-conversion efficiencies (PCEs). The maximum high PCE reaches up to 7.22% with a bulk-heterojunction (BHJ) thickness of 320 nm. This high efficiency was obtained by eliminating solvent additives such as 1,8-diiodooctane (DIO) to find an alternative way to control the domain sizes in the BHJ layer. Furthermore, the generalized transfer matrix method (GTMM) analysis has been applied to confirm the effects of applying a different thickness of BHJs for organic solar cells from 100 to 320 nm, respectively. Finally, the study showed an alternative way to achieve high PCE organic solar cells without additive solvent treatments to control the morphology of the bulk-heterojunction. During the last decades, bulk-heterojunction (BHJ) small-molecule organic solar cells (OSCs) have received more research attention ,6,7,8,9.i.e., chlorobenzene, 1,2-dichlorobenzene) -phenyl-C71-butyric-acid-methyl-ester (PC70BM) to form a BHJ photoactive layer. Chemical structures for OSC showed in p-DTS(FBTTh2)2 and PC70BM were dissolved in chlorobenzene (CB) and stirred over 24 h with a total concentration of 50 mg/mL. Indium tin oxide (ITO)-coated glass substrates were cleaned sequentially by ultrasonic treatment in Alconox detergent, deionized water, acetone and isopropyl alcohol. A polymeric conducting thin layer of PEDOT:PSS (40 nm) was spun-cast on top of the ITO-coated glass substrate. Before spin-coating of BHJ thin film, co-dissolved donor-acceptor blend was heated at 100 °C for 30 min. Then the p-DTS(FBTTh2)2:PC70BM BHJ layer was spin-cast from the heated and blended solution with different spin speed to form a different thickness of BHJs from 300 to 2000 rpm. During the solution preparation and film formation processes, 1,8-diiodooctane (DIO) additive solvent treatment was avoided. Finally, Ca (5 nm) and Al (100 nm) electrodes were deposited on top of BHJ photoactive layer  characterization system was supplied by PV Measurement Inc.  to obtain the data. A GTMM analysis was accomplished to calculate and analyse the multi-layered interface OSCs. For transfer matrix analysis, optical constants of all layers have been obtained by the ellipsometry method. Finally, the nano-morphology of BHJ film was investigated by atomic force microscopy (AFM).To address the particular issues mentioned above, OSCs fabricated through the incorporation of a donor-acceptor blend of ve layer b. Post-a2 Air Mass (AM) 1.5G illumination. The optimized ratio of the small molecule donor (p-DTS(FBTTh2)2) to PC70BM was chosen to be the same as the reported ratio (60:40 w/w) [J-V) characteristics of SM BHJ OSCs, in terms of BHJ without DIO treatment, with different photoactive thicknesses are shown in SM BHJ OSCs have been tested under simulated 100 mW/cmVoc as high as 0.74 V was observed in all devices. Combined with its high Jsc and fill factor (FF), a high power conversion efficiency (PCE) of 6.83% on average was measured with 320-nm-thick devices and the highest PCE was measured at 7.22% . The measured PCE of our device (2.78%) is much greater than the reported PCE value (1.8%) with the same BHJ thickness of 100 nm [p-DTS(FBTTh2)2 and small molecule donor materials.A J-V result. The results are shown in Jsc value by integrating the EQE data. The calculated Jsc values are in good agreement with the directly measured values. Furthermore, these EQE spectra confirm Beer-Lambert law by increasing the photoactive layer thicknesses. It is good to note that EQE spectra results obtained much better results to compare with the literature [The EQE spectra were obtained to confirm the accuracy of the SM BHJ OSC’s photo-generated terature ,3,4,8. A−1∙cm−3) of SM BHJ OSCs obtained by the generalized transfer matrix method (GTMM). These calculated charge generation rate results provide a further explanation as to why a thicker junction of OSC has a higher Jsc value than a thinner junction of OSCs. From Jsc value in thin junction OSCs. By increasing the photoactive layer thickness containing BHJ, the OSC follows Beer-Lambert law. Therefore, the photo-generation rate is exponentially decreased by increasing the photoactive layer thickness.p-DTS(FBTTh2)2:PC70BM BHJ film (thickness = 320 nm) is shown in An AFM two-dimensional 2D) topography image of the D topograIn conclusion, we studied the high performance of small-molecule organic solar cells without additive solvent treatment on the control of the photoactive layer in nano-morphology. Significantly, we have obtained small-molecule organic solar cells with a comparable PCE of 7.22% by eliminating the DIO additional solvent treatment. Also, GTMM analysis was accomplished to confirm the effect of the thickness variations of SM BHJ OSCs. Lastly, AFM measurement also confirms that smaller domains have achieved efficient charge generation. These results provide significant progress in showing that solution-processed small-molecule organic solar cells without solvent treatment can be an alternative method to having a high-PCE device with polymeric and/or small-molecule counterparts.


The Uncoordinated 5A (UNC5A) protein is part of a family of receptors that play roles in axonal pathfinding and cell migration. We previously showed that the Fanconi anemia C protein (FANCC) interacts with UNC5A and delays UNC5A-mediated apoptosis. FANCC is a predominantly cytoplasmic protein that has multiple functions including DNA damage signaling, oxygen radical metabolism, signal transduction, transcriptional regulation and apoptosis. Given the direct interaction between FANCC and UNC5A and that FANCC interferes with UNC5A-mediated apoptosis, we explored the possibility that FANCC might play a role in axonal-like growth processes.Here we show that FANCC and UNC5A are localized to regions of neurite outgrowth during neuronal cell differentiation. We also show that absence of FANCC is required for neurite outgrowth. In addition, FANCC seems required for UNC5A expression. Results from this study combined with our previous report suggest that FANCC plays a role in tissue development through the regulation of UNC5A-mediated functions. FancA and FancC are highly expressed in the developing brain, specifically in the intermediate zone, which contains migrating neurons . Thase (AD) .UNC5A were found in the posterior cingulate brain region of AD patients, while mutations in UNC5C seemed to predispose to late-onset Alzheimer’s disease [Furthermore, significant expression changes in  disease –31. ThesIt is unclear whether interaction between FANCC and UNC5A is required for neurite outgrowth. Further work is needed to determine whether FANCC regulates UNC5A apoptosis during cellular development or axon guidance in vivo.


The adverse drug reactions (ADRs) related to clonazepam are mild, and only two cases of myotoxicity induced by clonazepam have been reported, with both patients recovering well. We present a unique case of a serious ADR outcome after taking clonazepam.A 24-year-old woman with a long-standing history of polio and a 2-year history of epilepsy developed a serious ADR after repeated exposure to oral clonazepam combined with sodium valproate that manifested as myotoxicity and elevated levels of creatine phosphokinase. The patient is currently bedridden and unable to take care of herself.Clinicians should be vigilant of the possibility of myotoxicity induced by clonazepam, especially in specific populations such as polio patients or when clonazepam is used in combination therapies. Epilepsy is one of the most common serious disorders of the brain, affecting about 50 million people worldwide. Nearly a quarter of these patients have drug-refractory epilepsy . ClonazeThere has been no previous report on serious adverse drug reactions (ADR) associated with clonazepam. Here we describe a patient who developed myotoxicity and became bedridden after repeated exposure to clonazepam combined with sodium valproate.A 24-year-old woman had experienced non-progressive polio from 1 year of age despite having been inoculated with polio vaccine on time. She could walk with a limp but could not control her right hand sufficiently well to write, and so had not received the usual schooling. However, she could take care of herself and help her parents with a small amount of housework. About 2 years previously she had suffered from epilepsy in the form of bilateral tonic-clonic seizures, but she had not received regular treatment due to financial reasons. The epilepsy had not resulted in any deterioration of her polio symptoms.The patient had been taken to the hospital to receive regular treatment for epilepsy for the first time 6 months previously, at which time her antiepileptic regimen was sustained-release sodium valproate (500 mg p.o.) plus clonazepam (2 mg p.o.) every 12 h. The patient developed drowsiness after the first combined dose of sodium valproate and clonazepam, and slept from 3 p.m. on the first day to 10 a.m. on the following day. She could not walk unaided and felt very tired, but she did not contact her doctor, instead continuing on this antiepileptic regimen regularly for 21 days until she ran out of clonazepam tablets. Her epilepsy symptoms were well controlled during this 21-day treatment period, but she developed muscle weakness and muscle pain, and remained in bed since she could not take care of herself.The patient continued taking the sustained-release sodium valproate tablets (500 mg p.o. every 12 h), during which her epilepsy symptoms remained well controlled. Her muscle weakness symptoms started to improve gradually over the following 2 weeks, allowing her to stand but not walk. At this time she was taken to hospital for the second time. The patient and her family unfortunately refused electromyography, a muscle biopsy and other tests with the exception of some simple blood tests due to financial reasons. The findings of preliminary blood examinations including the complete blood count, urine test, liver and kidney function test, electrolytes, and plasma ammonia were within clinically acceptable limits, but her serum CPK was markedly raised at 4261 U/L (38–174 U/L), while her serum sodium valproate concentration was 101.89 μg/mL (50–100 μg/mL) and her serum globulin concentration was 36.4 g/L (25–35 g/L). There had been no preceding illness, infection or trauma, the patient did not have a history of statins or other drugs, and there was no family history of any neuromuscular disorder. Based on the relationship between medication times and symptoms, we attributed the myotoxicity to clonazepam, and assigned the patient to an antiepileptic regimen of sodium valproate monotherapy, with clonazepam remaining discontinued.A 2-month telephone-based follow-up revealed that the patient had started taking clonazepam irregularly because of insomnia, and suffered from muscle weakness and muscle pain again. Her clinical condition had deteriorated to the point that she was unable to stand or walk, and was unable to take care of herself. The patient and her family refused further physical examinations and treatment because they had lost confidence in curing the disease and also for financial reasons. We recommended that the patient discontinued clonazepam immediately and never take it again. At another follow-up 3 months later, the frequency and severity of epileptic seizures were significantly reduced in the patient, but her myotoxicity condition had not improved, she still could not stand or walk, and she was now bedridden.Our patient developed muscle weakness after taking sodium valproate and clonazepam at the same time, and the myopathy caused by sodium valproate and by clonazepam had been reported –9. HowevThe patient and her family declared that her polio and epilepsy were non-progressive. However, the dearth of neurological examinations meant that the woman might have actually had a slowly progressive early-onset type of muscle-wasting disease of neurological or myopathic origin, whose acceleration by the epilepsy treatment could have caused her clinical manifestations. Moreover, the probable myopathy varied with her clonazepam intake, representing further evidence that clonazepam was related to the myotoxicity. Numerous agents exhibit well-documented myocytoxicity, including anticholesterol statins, antirheumatic/inflammatory/immunosuppressive drugs, antinucleoside analogues, contaminated products, and dietary agents, and they result in symptomatologies ranging from mild discomfort and inconvenience to permanent damage and disability , 12. TheBecause our patient was taking sodium valproate at the same time as clonazepam, drug interactions might have been present. Jeavons et al.  reportedThe reported myocytoxicity symptoms including ataxia, fatigue and weakness induced by clonazepam were resolved and the CPK level reduced to the normal reference range after stopping clonazepam in our patient , 6. The We have described a case of the serious ADR of myotoxicity that was probably induced by clonazepam. Our findings suggest that clinicians should be vigilant of the possibility of myotoxicity induced by clonazepam, especially in specific populations such as polio patients or when clonazepam is used in combination therapies.


Subsequently, the synthesized compounds have been evaluated for their antimalarial activity against the drug-sensitive P. falciparum NF54 strain. All of them were inactive. In addition, they did not show any toxicity against L6 cells . These results contribute to a better understanding of artemisinins mechanism of action.Herein, we describe a biomimetic entry to (+)-3-hydroxymethylartemisinin ( Gratify9 and 10 (12 and 13 after treatment with Martin sulfurane [12 using NiCl2/NaBH4 in methanol as solvent yielded derivative 14 with excellent diastereomeric ratio (dr = 1:0.03). On the other hand, derivative 13 was reduced under Birch conditions (Li/NH3) to afford ester 15 in 61% yield (dr = 1:0.4). Gratifyingly, both artemisinin precursors 14 and 15 possess the desired stereoconfiguration at carbon center 8a.The structure of all these isomers was confirmed by NOE experiments  in 24% and 16% yield, respectively. Protection of the free hydroxy group of 16 as a silyl ether and methylation of the obtained lactone in α-position (LDA/MeI/HMPA) afforded derivative 17. After removal of the TES protecting group (+)-3-hydroxymethyl-9-epi-artemisinin  was produced from 17 in two steps including epimerization and cleavage of the TES group in excellent yield  as well as of the derivatives 16 and 18 against the drug-sensitive P. falciparum NF54 strain as described previously [2 did not show any toxicity against L6 cells . In both assays the highest concentration used was 100 μg/mL.Finally, we evaluated the antimalarial activity of (+)-3-hydroxymethylartemisinin  [2+ in vitro (Fenton reaction), it is difficult for us to explain their inactivity against Plasmodium. Furthermore, in cellular systems free ferrous iron [Plasmodium parasite. Derivative 2 will be used to attach further substituents at position 14 of artemisinin to prove this hypothesis.In the past it has been postulated that artemisinins kill intraerythrocytic parasites such as es (ROS) . As arteous iron  as well ous iron –21 have ous iron ,22. Our Experimental details, NMR spectra and other physical data are shown in File 1Experimental part.


Giant cell tumor of bone (GCTB) is a biologically benign and locally aggressive tumor that most often affects the epiphyseal and metaphyseal sites of long bones in the young adult population. Overexpression of receptor activator of nuclear factor kappa B ligand (RANKL) by cancerous mesenchymal stromal cells stimulates a signal transduction cascade that recruits and activates multinucleated osteoclast-like giant cells, resulting in pathologic bone resorption. Denosumab, an RANKL inhibitor that blocks the RANKL-mediated osteoclast activation, has been recently approved by the United States Food and Drug Administration (FDA) for the treatment of aggressive GCTB. Although uncommon, several studies reported drug-related malignant morphological transformation of benign GCTB following treatment with denosumab therapy. The aim of the article was to review the clinicopathological characteristics of all the reported cases of malignant sarcomatous transformation of GCTB after treatment with denosumab therapy in patients without any history of prior exposure to radiotherapy. Giant cell tumor of bone (GCTB) is a benign osteolytic neoplasm that primarily involves the epiphyseal and metaphyseal regions of long bones . AlthougThe optimal management of GCTB continues to be controversial . NonetheFrom a histological point of view, GCTB has a distinctive bi-phenotypic cell pathology. GCTB comprises osteoclast-like multinucleated giant cells that express receptor activator of nuclear factor kappa B (RANK) and neoplastic mesenchymal stromal cells that express RANK ligand (RANKL). In patients with GCTB, the RANK–RANKL interaction mediates the differentiation and activation of osteoclasts and thus results in the osteolytic phenotype of bone destruction ,6-8.Denosumab is a fully humanized monoclonal antibody that targets RANKL. Thus, denosumab inhibits the RANK–RANKL interaction and prevents bone destruction . In mid-Several studies including clinical trials ,10-12, cPrior exposure to radiation therapy has been proposed as a predisposing factor for the development of malignant transformation of GCTB . AlthougPubMed® database was searched until 15th December 2018 using the following keywords: “giant cell tumor of bone”, “denosumab”, and “malignant transformation”. Only English-published literature was included. Further references from published articles were also manually screened for potential additional studies. The study inclusion criteria included patients who were (i) initially diagnosed with benign GCTB, (ii) received denosumab therapy, (iii) developed malignant sarcomatous transformation during or post-therapy, and (iv) did not have any history of previous exposure to radiation therapy. For each reported case of sarcomatous transformation of GCTB during or after denosumab therapy, the following details were reported (whenever available): authors, year of publication, patient age, patient gender, tumor site, sarcoma histology, pulmonary metastases, causal relationship with denosumab therapy, time of sarcomatous transformation from the time of denosumab therapy, and duration of denosumab therapy.In 2010, Thomas et al.  reportedIn 2013, Chawla and colleagues  reportedIn 2015, Rutkowski and associates  reportedIn 2015, Aponte-Tinao and partners  reportedIn 2015, Broehm and friends  reportedIn 2016, Park and peers  reportedIn 2017, Tsukamoto and co-workers  reportedTable The exact mechanism by which the sarcomatous transformation of GCTB occurs during or after denosumab therapy is poorly defined . HoweverFurthermore, from a technical point of view, a potential hypothesis has been proposed to account for the sarcomatous transformation of GCTB that takes place during or after denosumab therapy. This hypothesis takes into account that osteosarcoma arising in a histologically benign GCTB may encompass some scattered foci of malignant tumor. These foci of malignant tumor (osteosarcoma) may have been missed by means of sampling error at the time of initial core biopsy ,40. MoreIn literature, apart from the sarcomatous transformation, a wide variety of morphological transformation of GCTB has been reported during and after denosumab therapy. For instance, some authors described growth patterns resembling osteoblastoma , fibrousAt the present time, the optimal duration of denosumab therapy is not yet established. In addition, whether the duration of denosumab therapy is directly associated with the increased potential of developing sarcomatous transformation of GCTB remains poorly defined. Nevertheless, long-term treatment with denosumab therapy should be limited to the minimum possible in order to avoid dose-dependent toxicity side effects, for example, osteonecrosis of the jaw .n = 11) may not represent the actual figure.Lastly, it should be noted that this review article may not be reporting all the cases of malignant sarcomatous transformation of GCTB, as under-reporting of such cases by healthcare centers remains a possibility. Thus, the number of related cases reported in this review (n = 11) related cases have been reported so far in the English literature. The exact mechanisms or triggering factors by which sarcomatous transformation of GCTB occurs during or after denosumab therapy are poorly defined. Additional isolated case reports and large-sized cohort studies are needed to thoroughly explore a causal association between the malignant sarcomatous transformation of GCTB and denosumab therapy.In conclusion, healthcare providers should be aware that malignant sarcomatous transformation of GCTB during or after denosumab therapy is a possible consequence. To the best of knowledge, 11 (


In both groups, the non-treated side served as a control. A numerical rating scale (NRS) of pain, pressure pain thresholds (PPTs), cold detection thresholds (CDTs), warmth detection thresholds (WDTs), cold pain thresholds (CPTs), and heat pain thresholds (HPTs) were tested on both sides at the gingiva and canine tooth and on the hand. The data were analysed by a repeated measures analysis of variance (ANOVA). The NRS pain scores were significantly lower in the LG group (P = 0.01). The CDTs, CPTs, WDTs, HPTs, and PPTs at the gingiva and the PPTs at the canine tooth were significantly less sensitive on the treatment side of the LG compared with that of the PG (P < 0.033). The parameters tested also showed significantly less sensitivity on the non-treatment side of the LG compared to that of the PG (P < 0.043). There were no differences between the groups for any quantitative sensory testing (QST) measures of the hand. The application of LLLT appears to reduce the pain and sensitivity of the tooth and gingiva associated with orthodontic treatment and may have contralateral effects within the trigeminal system but no generalized QST effects. Thus, the present study indicated a significant analgesia effect of LLLT application during orthodontic treatment. Further clinical applications are suggested.Low-level laser therapy (LLLT) may have an effect on the pain associated with orthodontic treatment. The aim of this study was to evaluate the effect of LLLT on pain and somatosensory sensitization induced by orthodontic treatment. Forty individuals  scheduled to receive orthodontic treatment were randomly divided into a laser group (LG) or a placebo group (PG) (1:1). The LG received LLLT  can reduce pain and sensitivity in teeth and surrounding tissues. They divided 40 patients into two groups; one was given repeated LLLT in the hours and days following procedures, while the other group received a placebo course. They tested both groups for sensitivity to stimuli including heat and pressure, in the mouth and on the hands. LLLT significantly reduced pain in the mouth relative to the placebo group. No differences were found in tests on the hands, suggesting LLLT works as a targeted analgesia. The loading and forces may lead to an inflammatory process in the periodontal ligament, which can cause pain.10 The pain usually begins within 4 h after the force is applied, reaches a peak after ~24 h, and then dissipates by day 7.12 The intensity of the pain during orthodontic treatment is sometimes reported to be even stronger than the pain related to dental extractions.13 Thereby, it could be one of the reasons for non-compliance with and even withdrawal from orthodontic treatment.The most common problems for patients during orthodontic treatment are pain and discomfort evoked by the appliances and mechanical loading.15 Overall, studies on clinical efficacy have shown equivocal results.16 The efficacy of LLLT in alleviating orthodontic pain has also been studied in recent years.22 It has been reported that LLLT has analgesic properties and anti-inflammatory effects25 through increases in the local blood flow, reduction of prostaglandin levels E2 and inhibition of cycloxygenase-2.24 Various studies have been designed to investigate the pain during orthodontic treatment, including telephone interviews and questionnaires.27 However, because of small sample sizes, controversial results, and different methodological issues in previous studies, good evidence for a positive treatment effect of LLLT is still lacking. Randomized, placebo-controlled and double-blinded studies are needed.28Low-level laser therapy (LLLT) has attracted attention for decades because of its obvious advantage in pain management as a non-invasive and inexpensive technique without significant adverse effects.29 QST in the trigeminal region has been characterized in terms of specificity, sensitivity, repeatability, and reliability.31 The application of QST could help to study the underlying neurobiological mechanisms of orthodontic pain and could serve as a valuable measure to determine the effects of LLLT on somatosensory function. One of our recent studies has shown that pressure pain thresholds (PPTs) applied to the teeth have excellent intra-examiner and inter-examiner agreements in healthy participants.32 A modified intra-oral QST has been applied in several of our previous studies34 and has been shown to be an easy and reliable technique for assessing mechanical pain sensitivity  in the periodontal ligament, which is associated with endodontic or periodontal conditions.Quantitative sensory testing (QST) is a psychophysical method in which different standardized stimulus modalities  are applied to different tissues , and the test person’s response, in terms of a sensory or pain threshold or a report of the magnitude of the perceived intensity, is assessed.As a measure of self-reported levels of pain, the numerical rating scale (NRS) score, in which 0 represents “no pain” and 10 indicates “the most pain imaginable”, can be recorded from the participants. The aim of the present study was to evaluate the effect of LLLT on the self-reported pain and somatosensory function induced by orthodontic treatment using our recently established intra-oral QST techniques in a randomized, placebo-controlled and double-blinded study design.P > 0.916). There were no significant differences between the LG and PG at baseline for cold detection threshold (CDT) , cold pain threshold (CPT) , warmth detection threshold (WDT) , heat pain threshold (HPT) , pressure pain threshold (PPT) at the gingiva , or pressure pain threshold (PPT) at the canine tooth .There were no significant age or gender differences between the laser group (LG) and placebo group (PG) at baseline . There was also a significant effect of time , with the highest NRS scores being recorded at 24 h (P = 0.018). However, there was no significant interaction between the groups and time  , side  and time  and a significant interaction between group, side, and time . Moreover, the interaction between group and time was also significant ; whereas, the interaction between side and time was not significant . The relative PPT changes were significantly larger in the LG compared with the PG at 2 h , 24 h , 4 d , and 7 d  at the treatment side and non-treatment side. When the treatment side in the LG was compared to the non-treatment side in the same group, there were significant effects of side  and time  but no significant interaction between side and time . The relative PPT changes on the treatment side were significantly higher than those on the non-treatment side from 2 h to 7 days in the LG , 24 h , 4d , and 7d . Finally, there was a significant interaction between group and time .Interestingly, the relative PPT changes at the non-treatment side in the LG were significantly higher compared with those at the non-treatment side in the PG at 2 h  and side (P = 0.243) but a significant effect of time (P < 0.001). However, when the non-treatment sides in the LG and PG were compared, there were significant effects of group  and time . The interaction between group and time was not significant   Fig. .The mean thermal QST values are shown in Table F = 9.2, df = 1, P = 0.003) and time  but no significant effect of side . The interaction between group, side and time was not significant , but the interaction between group and time was significant . The relative CDT changes were significantly smaller in the LG than in the PG at 2 h , 24 h , 4 d , and 7 d   Fig. . The relF = 7.2, df = 1, P = 0.009) and time  but no significant effect of side . The interaction between group, side and time was not significant , but the interaction between group and time was significant . The relative WDT changes were significantly smaller in the LG than in the PG at 2 h , 24 h , and 4 d   Fig. . The relF = 28, df = 1, P < 0.001) and time  but no significant effect of side . The interaction between group, side, and time was not significant , but the interaction between group and time was significant . The relative CPT changes were significantly smaller in the LG than in the PG at 2 h , 24 h , 4 d , and 7 d  . The relative CPT changes were also significantly lower on the non-treatment side than on the treatment side in the PG at 2 h, 24 h, 4 d and 7 d   Fig. . The rel01) Fig. .F = 7.1, df = 1, P = 0.009) and time  but no significant effect of side . The interaction between group, side and time was not significant . The relative HPT changes were significantly larger in the LG than in the PG from 24 h to 7 d , WDT , CPT , HPT  and PPT . There were significant time effects for CDT , WDT , CPT , and HPT , but there was no time effect for PPT  , ATP production, and prostaglandin reduction.41 In addition, LLLT might alter nerve conduction by influencing the synthesis, release, and metabolism of encephalin, endorphins and many other neurochemicals.42 This study was not designed to elucidate the neurobiological mechanisms potentially underlying an analgesic effect of LLLT on orthodontic pain, but the present findings so far indicate that the observed reduction in the NRS pain scores cannot be attributed to placebo-based mechanisms. Although placebo effects cannot be completely ruled out based on the self-reported NRS pain scores, the observation of consistent and significant effects on somatosensory function further supports the notion of a neurobiological effect of LLLT.The exact mechanisms responsible for the apparent analgesic effect of LLLT are still unclear.33 To our knowledge, it was the first time that QST was applied to evaluate the effect of LLLT on orthodontic pain in a systematic manner. The QST parameters are considered to offer a diagnostic sensitivity of 67%–100% for small-fiber neuropathy and to be potentially valuable for evaluating small-fiber function.44 The CDT is considered to represent small myelinated Aδ nerve fiber conduction, the WDT is considered to represent unmyelinated C nerve fiber conduction, and the CPT and HPT may represent conduction by both Aδ and C nerve fibers.46 The PPT was measured to test deep pain sensitivity, which is probably mediated through both C and Aδ fibers.47 The PPT can also be used to assess mechanical pain sensitivity in the periodontal ligament and gingiva.32As a special feature of the present study, intra-oral QST was applied in addition to traditional self-reports of pain intensity evoked by the orthodontic treatment. QST is believed to contribute to a better understanding and profiling of the underlying neurobiological mechanisms related to orthodontic pain.The present QST results demonstrated that not only tooth-related pain but also sensitization of thermal and mechanical somatosensory channels were evoked by the orthodontic treatment. In fact, both mechanical (PPT) sensitization and thermal  sensitization were observed during the orthodontic treatment until day 7. In the present study, all of the QST parameters were normalized to their baseline values (0 h), and the relative changes at the 5 time points in the two treatment groups were calculated and presented Figs.  and 3. I32 The force from the pressure algometer was applied directly to the crown of the canine tooth, and the recorded PPT may partially reflect the innervation patterns and receptor density in the periodontal ligament.48 Interestingly, in the present study, significantly higher PPTs  on the LLLT-treated side compared to the non-treated side were observed in the LG. Thus, it can be speculated that LLLT may decrease the sensitization of the periodontal ligament evoked by orthodontic treatment. Surprisingly, the analgesic effect induced by LLLT was not restricted to the treatment side and was also observed on the contralateral side, indicating an extended effect in the trigeminal area. Studies on animals have shown the existence of branched nerves innervating both intrapulpal and periodontal tissues.49 It has also been shown in animal studies that LLLT may promote neural regeneration,37 but until now, only few animal studies have been available that allow for the study of the activation of the trigeminal-vascular system. Within the meninges, the activation of the trigeminal-vascular system  leads to an ipsilateral inflammatory response characterized by vasodilation.50 Animal studies have also indicated that noxious stimulation may produce marked blood flow changes in various orofacial structures.51 Noxious stimulation of the human teeth is indeed associated with bilateral increases in blood flow in both the maxillary and mandibular nerve innervation territories.52 In the trigeminal-vascular system in rats, prostaglandin E2 (PGE2) is considered to be a key mediator for pain and nocifensive responses.53 PGE2 causes vasoconstriction and is increased in both blood and saliva during migraine attacks, which is a mechanism related to pain. Interestingly, PGE2 may be reduced by LLLT,13 which is related to the activation of the trigeminal-vascular system, which, again, might be associated with the bilateral effects of LLLT.Mechanical allodynia in the periodontal ligament around the canine tooth was investigated with the same method used in our previous study.54 and lead to an increase in the intrapulpal pressure.55 Thus, increased fluid flow from the root canal system through the apical foramen and dentinal tubules may cause the delivery of SP and MMP-8 to the periodontal ligament space.56 These data support the possibility for the local neurogenic spread of inflammation from intrapulpal tissues to surrounding periodontal tissues, which may further explain the bilateral effect of LLLT.Tooth pain caused by orthodontic tooth movement is associated with elevated gingival crevice fluid contents of matrix metalloproteinase (MMP)-8 and neuropeptide substance P (SP) levels, which are more abundant in pulp tissues from painful human teeth33Pain only evoked local elevations in the gingival crevice fluid content of SP and MMP-8 levels and caused no marked modulations in systemic cardiovascular parameters. Thus, systemic stress mechanisms probably did not significantly contribute to the present results, which may explain the lack of significant QST effects on the hand. Time effects of the QST parameters at control sites, such as the hand, have also been observed in a previous study and may be attributed to minor drifts in psychophysical performance, e.g., an adaptation or a habituation of the test procedures.57 The evidence suggests that LLLT can be effective in enhancing the rate of orthodontic tooth movement since LLLT is found to increase the rate of bone remodeling without imposing any adverse effects.59 However, other studies have reported conflicting results60 and have failed to observe any significant improvement in the rate of orthodontic tooth movement associated with LLLT.61 The rate of tooth movement was not recorded in the present study, and further studies will be needed to clarify the effect that LLLT has on tooth movements.The process of bone remodeling in periodontal tissues is a major determinant of orthodontic tooth movement.62 Nevertheless, the strength of the mixed study group is that the present results may be more generalizable. Another limitation of the present study could be argued to be the subjective nature of the self-reported pain. However, in addition to self-reports of pain, we also included psychophysical responses (QST), which also indicated significant effects on the treatment side in the LG. In future studies, objective measures of somatosensory function  and nociceptive activity  could be incorporated into the study design to further support the potential analgesic effect of LLLT in relation to orthodontic pain. A particular strength of the present study was its double-blinded and placebo-controlled design. However, a limitation may still be that no information was collected to demonstrate that participants were indeed successfully blinded and that participants in the PG had the same degree of anticipation and expectancy of a positive outcome. Previous studies have clearly demonstrated that expectancy reports are crucial for the understanding of placebo effects.63 Thus, it may not be possible to completely rule out that the present findings were not contaminated by placebo effects. However, the QST findings may nevertheless suggest that there could be physiological responses related only to LLLT. Further studies are obviously needed to address these questions in more detail.One of the limitations of the present study could be argued to be the mixed group of participants, which includes both children and adults and includes participants of both genders. A total of 10 individuals were under 18 years old, but so far, there is no systematic information about age-related differences in acute pain responses evoked by orthodontic treatment, although gender differences in pain sensitivity have been documented.The volunteers were recruited from the orthodontic clinic at the Hospital of Stomatology of Nanjing Medical University . The inclusion criteria were individuals who were going to start comprehensive orthodontic treatment for slight crowding without tooth extraction, and all of the participants were to be healthy without signs or symptoms of pain or any kinds of on-going therapy and no other systemic disease. The exclusion criteria were active caries, periodontal diseases, visible lesions of the oral mucosa, and any kind of chronic use of analgesics or drugs affecting the function of the central nervous system.20 All of the participants gave informed consent to the procedures, which were approved by the local Ethical Committee  in accordance with the Helsinki Declaration II. In addition, all of the participants understood that they were free to withdraw from the experiment at any time.Fifty participants requiring orthodontic treatment were initially recruited in this study: six participants declined enrollment after receiving study information, and an additional four individuals were excluded according to the exclusion criteria. A total of 40 participants  were included in the study  by Examiner 2. The LG received the low-level laser therapy (LLLT) on one randomized side from Examiner 2 immediately after (0 h) the arch wire (0.014 super plastic nickel-titanium arch wire) was placed in self-ligating brackets  and then again at 2 h, 24 h, 4 d, and 7 d. The PG received inactive treatment with the LLLT device, which was applied in a similar way at the same test sites and at the same time points as for the LG. The treatment sides in the LG and PG groups were also randomly determined using the same computer program by Examiner 2. QST was performed after the application of active LLLT or placebo LLLT in both of the groups. A 0–10 numerical rating scale (NRS) score, in which 0 represented “no pain” and 10 indicated “the most pain imaginable”, was recorded from the participants, and the modified intra-oral QST was performed at the canine tooth and the surrounding gingiva on both sides at 0 h, 2 h, 24 h, 4 d, and 7 d after the orthodontic forces were applied. The participants were requested to close their eyes during the LLLT application. The study was performed in a quiet and temperature-controlled room. The contralateral, non-treatment side served as an additional control in both groups. Examiner 1 was blinded to the information about the allocation to the LG or PG, as well as to which side had been treated or not.The experiment was performed as a randomized, placebo-controlled and double-blinded trial. All of the participants were tested by the same examiner (Examiner 1: Song Wu). The laser treatment and randomization were performed by another examiner (Examiner 2: Huijie Shen). The forty participants were randomly divided into two experimental groups: a LG and a PG. The randomization code of the two treatment groups (20 in each group) for the 40 participants was obtained using a computer program  or when the temperature reached a sensation of painful hot or cold (CPT and HPT). The temperature of the thermode returned to the baseline after the mouse button had been pushed. The range of the stimulation temperatures was between 0 °C and 50 °C. The CDT and the WDT were always measured first, followed by the CPT and the HPT. The mean of three repeated trials was used to determine the threshold values.Thermal quantitative sensory tests were performed using a computerized thermal stimulator . The contact area of the intra-oral thermode was 6 mm× 6 mm. The cold detection threshold (CDT), warmth detection threshold (WDT), cold pain threshold (CPT) and heat pain threshold (HPT) were tested at the area of the gingiva around the upper-left and right canines and on the left hand  at the five time points. The thermode started from a baseline temperature of 37 °C. The temperature decreased or increased by 1 °C·s33 The pressure was increased with a constant application rate of 30 kPa·s-1. The participants were instructed to concentrate on the test stimulus and to press the switch button as soon as they felt that the pressure changed to the slightest sensation of pain. The amount of pressure (kPa) at this point was defined as the PPT. Three measurements per site were made at 1-min intervals to obtain a mean value.The mechanical PPT was measured by an algometer . The diameter of the intra-oral probe was 8 mm. The algometer was applied perpendicular to the surface of the test sites. The measuring sequence was the left hand, upper-left canine gingiva, upper-right canine gingiva, upper-left canine, and upper-right canine.-2 .LLLT was applied buccally and lingually to an upper canine at 6 points: mesial, distal, and at a site corresponding to the middle of the root of the canine tooth for 20 s each in the LG. The laser tip diameter was 1 mm, and the laser tip was kept 10 mm away from the surface of the gingiva during the stimulation. From the cementoenamel junction of the tooth towards the apex of the tooth, the laser tip was directed perpendicular to the long axis of the tooth. In the PG, the LLLT device was held at the same 6 points for the same duration with the light on for the operation indicator. The same procedure was repeated for the LG but without any active laser output. Both the examiner and the participant wore protective laser glasses. LLLT was applied using an 810-nm gallium-aluminium-arsenic diode laser in continuous mode with the power set at 400 mW, 2 J·cmThe sample size was calculated with risks for type I and type II errors of 5% and 20%, respectively, an estimate of the inter-individual variation of 25% and a minimal relevant difference for detection of 20%. A total of 40 patients were recruited in the present study.Descriptive statistics were used to summarize the data. Baseline comparisons between the two groups in regards to age, gender and the QST parameters at baseline were performed. The necessary logarithmic transformation was performed when the data were not normally distributed. The mean values and SD of the NRS score, CDT, WDT, CPT, HPT, and PPT at each time point and test site were calculated. All of the data were normalized to the baseline, and the relative changes of each parameter were compared between the treatment groups. A three-way mixed model analysis of variance (ANOVA) with repeated measures was used to analyse the different outcome parameters of the CDT, WDT, CPT, HPT, and PPT at the test sites at different time points. The between group factor in the ANOVA was group (LG and PG), while the within-group factors were side (treatment and non-treatment side), and time point . Post hoc tests were performed with the LSD honestly significant difference test with corrections for multiple comparisons. The significance level was set at 0.05. Blinding of the data was maintained in the statistical analysis.The QST data that support the findings of this study are available in figshare with the identifier 10.6084/m9.figshare.5532226.v1.A repeated application of LLLT using a gallium-aluminium-arsenic diode laser with an 810-nm wavelength was able to significantly reduce self-reported pain scores and sensitization of the periodontal and gingival tissues evoked by orthodontic treatment.The effect also extended to the contralateral side in the trigeminal region but not in the extra-trigeminal region, indicating that LLLT treatment may have some degree of bilateral effects within the orofacial region. This interesting finding calls for future studies on the clinical application of LLLT with larger cohorts, as well as for those including additional measures of nociceptive function and sensitization.


FN1, FLNA, FLNB, VCL, GSN, MYH10, ACTN4, KDR and EREG, and they were all down-regulated after 6-TG treatment. The coexpression network consisted of 18 microRNAs (miRNAs), 9 long noncoding RNAs (lncRNAs) and 20 mRNAs. Hsa-mir-16-5p and Hsa-mir-335-5p targeted the greatest number of mRNAs in the network. These molecules could bind to PAX8-AS1 and eliminate the inhibition of target mRNA expression. We showed that PAX8-AS1 is the main lncRNA affected by 6-TG and that PAX8-AS1 regulates the hub genes in tumor pathways by competitively binding with miR-16-5p and miR-335-5p.Breast cancer is one of the most prevalent and recurring cancer types that leads to deaths in women. Triple-negative breast cancer (TNBC) is difficult to treat due to the lack of therapeutic targets. Many studies have focused on identifying drugs for use as alternative treatments for breast cancer. Thioguanine (6-TG) exerts antitumor effects in cancer. Increasing evidence has demonstrated that competitive endogenous ribonucleic acids (ceRNAs) are involved in cancer processes. However, the mechanism by which 6-TG regulates lncRNA–miRNA–mRNAs has not been elucidated. We evaluated the antitumor effect of 6-TG in MDA-MB-231 cells and comprehensively analyzed the RNA-Seq data of MDA-MB-231 cells treated with 6-TG. Our results showed that most tumor pathways were blocked by 6-TG. The hub genes were  Breast cancer is one of the common causes of deaths in women. Among all diagnosed cases of invasive breast cancer, approximately 15–20% are triple-negative breast cancer (TNBC) .DNMT1 [2-M arrest and cell death by DNA mismatch repair mediated in human colorectal carcinoma. Our previous studies have shown that 6-TG can induce FAS-mediated exogenous apoptosis and p21-dependent G2/M arrest by restoring TP53 activity in MCF-7 breast cancer cells [Thioguanine 6-TG) is a classic leukemia therapy drug with potential for cancer therapy . 6-TG acDNMT1 . Inki et-TG is a DNMT1  suggesteer cells .BCRT1 is overexpressed in breast cancer and promotes breast cancer metastasis by targeting miR-1303 [NKILA suppresses TGF-β-induced epithelial–mesenchymal transition by blocking NF-KB signaling in breast cancer. Therefore, the theory of competitive endogenous ribonucleic acid (ceRNA) has emerged, which states that lncRNAs can competitively combine with the miRNA response element (MRE) and inhibit the negative regulation of target mRNAs by miRNAs. The ceRNA network has been confirmed in many diseases. However, the role of ceRNA in the regulation of breast cancer remains to be further studied.Intensive research in the past two decades has uncovered the presence and importance of noncoding RNAs (ncRNAs), which include microRNAs (miRNAs) and long ncRNAs (lncRNAs) . LncRNAsmiR-1303 . Wu et amiR-1303  reportedIn our study, we found that 6-TG plays an antitumor role in TNBC cells (MDA-MB-231 cells). To elucidate the interactive mechanism of lncRNAs and the lncRNA-mediated regulatory network, we performed an integrative analysis to identify differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) of TNBC cells after treatment with 6-TG and identified a ceRNA network that can down-regulate core genes that dominate the tumorigenesis process. The present study contributes to the exploration of the ceRNA regulatory mechanism of 6-TG and provides valuable insight for further functional research.2 at 37°C. The MDA-MB-231 cell line has been authenticated using STR profiling within the last 3 years. All experiments were performed with mycoplasma-free cells.The human breast cancer cell line MDA-MB-231 was purchased from the Cell Bank of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Cells were cultured in RPMI 1640 medium  supplemented with 10% fetal calf serum, 100 IU/ml penicillin and 100 μg/ml streptomycin . The cells were cultured in a humid environment with 5% CO6-TG was purchased from Selleck Chemicals . Dimethyl sulfoxide (DMSO) was purchased from Sigma–Aldrich .3 cells were seeded per well in a 96-well plate and treated with 6-TG at 0.5, 1, 2, 4 and 8 μM and DMSO. Thereafter, CCK-8 solution was added, and the absorbance was measured using a microplate reader  at 450 nm.Cells were treated with different concentrations of 6-TG and then subjected to the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s protocol . Briefly, 6.5 × 10Total RNA was extracted with TRIzol  reagent following the manufacturer’s instructions. The insert size was assessed using the Agilent Bioanalyzer 2100 system, and qualified insert sizes were accurately quantified using the StepOnePlus™ Real-Time PCR System . Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample (GEO number: GSE137418).http://bioinformatics.sdstate.edu/idep/). Genes with false discovery rates (FDRs) ≤ 0.1, |log2FC| ≥ 1.5 and P-value ≤ 0.01 were selected as candidate genes. Heatmaps were utilized to identify the gene expression differences between the control group and the 6-TG group. DEmRNAs and DElncRNAs are shown in Supplementary Tables S1 and S2, respectively.DEmRNAs and DElncRNAs were identified by the IDEP website (http://www.genome.jp/kegg/) database. The volcano plot of GSEA was structured on the website . Hierarchical clustering and heatmap plot analyses were performed for the ten main down-regulated pathways via the METASCAPE website (https://metascape.org/gp/index.html#/main/step1). The genes in the top ten pathways and seven pathways are shown in Supplementary Tables S3 and S4, respectively.Pathway analysis was used to investigate the DEmRNAs according to the Kyoto Encyclopedia of Genes and Genomes  network of genes involved in the main pathways was constructed to understand the relationships of different genes. The PPI network was constructed using Cytoscape3.7.2. The submodule of the PPI network was analyzed by using the MCODE plugin of Cytoscape. GO and KEGG pathway enrichment analyses for the DEGs involved in the module were performed.http://starbase.sysu.edu.cn/) and miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/). Correlation analysis was performed to evaluate the interaction between DEmRNAs and DElncRNAs. Pairs with absolute values of Pearson correlation coefficients not less than 0.95 were selected. The miRNAs of these lncRNAs were predicted by DIANA tools (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php).DEmRNAs in the MCODEs were collected for further analysis. Then, miRNAs of mRNAs were predicted from StarBase (https://echarts.apache.org/examples/zh/editor.html?c=sankey-simple).According to the ceRNA hypothesis, there were potential interactions among lncRNAs, mRNAs and miRNAs. We constructed the ceRNA coregulated network of DElncRNAs, DEmiRNAs and DEmRNAs using echarts. In brief, we used TarBase and miRTarBase to predict 18 miRNAs of 21 hub DEmRNAs. We also predicted coexpressed lncRNAs of the 21 hub genes according to Pearson correlation coefficients ≥ 0.95. Then, we utilized DIANA tools to predict the coexpressed miRNAs of the abovementioned DElncRNAs. Finally, we constructed a ceRNA network according to Supplementary Table S5 on the echarts website  reagent following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  . qPCR was performed with FastStart Essential DNA Green Master  via a StepOnePlus Real-Time PCR system. Primers sequences are listed in Supplementary Table S6. The results were analyzed using the 2https://www.oncolnc.org) and Kaplan–Meier plotter  to perform Kaplan–Meier survival analyses of the association between the expression of DElncRNAs, DEmiRNAs and DEmRNAs in the ceRNA network and the prognosis of patients. We only selected the TNBC sample set, which is composed of 255 patients with ER negative, PR negative, and HER-2 negative.To evaluate the potential roles of genes in the ceRNA network in the patients, we used Cox regression analysis of survival packages and Kaplan–Meier curves on OncoLnc . **50 of 6-TG in 48 h was 2.5 μM. Moreover, the cell morphology was changed after 6-TG treatment , Hsa-miR-1 (connection degree = 7) and Hsa-miR-335 (connection degree = 6) were the top three miRNAs that targeted most mRNAs, suggesting that they were major miRNAs in the coexpression network. Only six lncRNAs  were identified in the network according to the Lnc2Cancer 3.0 database. Therefore, PAX8-AS1 targeted the top two miRNAs and interacted with the majority of mRNAs ITGA2, LAMC1, ITGB4, KDR, FLNA, ACTN4 and EREG. To verify our analysis, the expression levels of the majority of mRNAs were assessed using qPCR. As shown in KDR, ITGA6, ACTN4 and EREG were significantly lower in the 6-TG group than in the control group. These data suggested that ceRNA played a potential role in regulating gene expression in MDA-MB-231 cells treated with 6-TG.To better understand the pivotal roles of DEmRNAs and DElncRNAs in DMA-MB-231 cells under 6-TG treatment, a ceRNA network was constructed. We used TarBase and miRTarBase to predict 18 miRNAs according to the abovementioned 21 hub DEmRNAs. We also predicted coexpressed lncRNAs of the 21 hub genes according to Pearson correlation coefficients ≥ 0.95. Then, we utilized DIANA tools to predict the coexpressed miRNAs of the abovementioned DElncRNAs. Echarts was used to construct the coexpression network. As shown in Hsa-miR-16 and Hsa-miR-335 seemed to exhibit protective functions, as the prognosis of patients with higher expression levels was longer than that of patients with lower expression levels. However, the ACTN4, EREG, FLNA and FLNB mRNAs  was reported as a regulator of cell proliferation, and its reduced expression could inhibit cell growth [Vash2 inhibited tumor growth by down-regulating EREG and IL11, suggesting the antitumor effects of EREG in tumors. Fortunately, our results showed that the tumor-related pathways, Regulation of actin cytoskeleton, MAPK signaling pathway, ECM–receptor interaction, Proteoglycans in cancer, Focal adhesion, PI3K-Akt signaling pathway and Pathway in cancer, were inhibited by 6-TG. In addition, we identified the hub genes in these pathways, such as FN1, FLNA, FLNB, ACTN4, VCL, GSN, MYH10, ACTG1, EREG and KDR, suggesting that they were the dominant genes in these pathways under 6-TG treatment.To the best of our knowledge, changes in a variety of cancer signaling pathways are involved in the development and progression of breast cancer. Previous studies have shown that MAPK signaling, the PI3K/AKT pathway and focal adhesion pathways play important roles in malignant transformation in TNBC ,18. KDR l growth . Yasuhirl growth  reportedCCND1, CCND3, CCNE1 and CDK [MiR-335 suppresses metastasis and migration by targeting the progenitor cell transcription factor SOX4 and has been identified as a metastasis suppressor miRNA in human breast cancer [miR-335-5p to regulate cell proliferation, apoptosis and invasion in pancreatic cancer. However, we did not observe any studies reporting lncRNAs with potential competing abilities. Increasing evidence indicates the crucial role of lncRNAs in the ceRNA network in terms of modulating other RNA transcripts [PAX8‐AS1 expression levels had shorter RFS times. While the isoform of PAX8-AS1 is named PAX8-AS1-N, it can bind to miR-17-5p and upregulate miR-17-5p targets, such as PTEN, CDKN1A, and ZBTB4. The reduced expression of PAX8-AS1-N indicated poor survival of breast cancer patients [PAX8-AS1 in ceRNAs is unknown. In our results, we found decreased lncRNA PAX8-AS1 expression under 6-TG treatment in MDA-MB-231 cells. The lncRNA PAX8-AS1 targeted the top two miRNAs and interacted with the majority of mRNAs in the ceRNA network. Moreover, the mRNAs with reduced expression after 6-TG treatment were consistent with those predicted by PAX8-AS1. Therefore, we predicted that PAX8-AS1 may sponge miR-16 and miR-335. This lncRNA can mediate ACTN4, EREG, KDR, FLNA, and FLNB expression by competing with miR- 16 and miR-335 to participate in the progression of MDA-MB-231 cells. Therefore, lncRNA PAX8-AS1 may inhibit MDA-MB-231 cells by sponging miR-16 and miR-335 to exert ceRNA regulation. Meanwhile, the high expression levels of miR-16 and miR-335 and the low expression levels of ACTN4, ERGE, FLNA, and FLNB could prolong patient survival, suggesting that 6-TG influenced the gene expression pattern and played a positive role in TNBC treatment.ceRNAs are transcripts that can regulate each other at the post-transcriptional level by competing for shared miRNAs . Amelia  and CDK . MiR-335t cancer . An et at cancer  found thnscripts . Wei et nscripts  reportedpatients . NeverthPAX8-AS1 is the main lncRNA influenced by 6-TG and that PAX8-AS1 regulates hub genes in tumor pathways by competitively binding with miR-16-5p and miR-335-5p. The present study contributes to the exploration of the ceRNA regulatory mechanism of 6-TG and provides valuable insight for further functional research.In summary, we showed that Click here for additional data file.


In February 2020, a novel coronavirus (SARS-COV2) broke out in Wuhan city of China. The Chinese government decisively imposed nationwide confinement. This study comprised a structured, online questionnaire, based on 40 items inquiring about socio-demographic information and anthropometric data (reporting weight and height), as well as changes in food intake, physical activity, and sleep during the COVID-19 outbreak. Questionnaires were distributed to residents of Jiangsu and other provinces from 29 March to 5 April. A total of 889 respondents were included, aged between 16 and 70 years . There was a significant increase in total food intake by 9.8% and a slight increase by 29.2% of respondents, and a significant decrease in physical activity by 31.5% and a slight decrease by 23.4% of respondents, especially in snacks and drinks, and outdoor activities. The rate of weight gain in the total population was 30.6% and the average weight gain was 0.5 ± 2.8 kg. The main factors contributing to weight gain were increased food intake and reduced physical activity. Additionally, normal-weight people were more likely to gain weight than people with overweight/obesity during the COVID-19 confinement. This study provided a good warning and educational reference value on lifestyle changes during the COVID-19 confinement. A novel coronavirus disease, later abbreviated to COVID-19 by World Health Organization (WHO) . It was As definitive vaccines and cures for COVID-19 are unlikely to be identified soon , social Nevertheless, the prolonged home life may unintentionally lead to increased sedentary behaviors, such as spending excessive amounts of time sitting, reclining, or lying down for screen activities , reducing regular physical activity (thus lowering energy expenditure), or indulging in excessive food intake that, consequently, causes an increased risk for and potential worsening of chronic health conditions . Some ofn = 1.097) found that almost 30% of subjects experienced weight gain (3.0 ± 1.6 kg), and overweight, obese, and older subjects tended to gain weight more frequently during COVID-19 lockdown [During the global pandemic COVID-19 lockdown, studies reported weight gain in different countries. For instance, the perception of weight gain was observed in 48.6% of the population in an Italian survey . A studylockdown .To comprehensively understand and analyze the impact of the domestic lifestyle due to the COVID-19 outbreak on the diet, exercise, and sleep of the Chinese people, we conducted this survey. It is worth mentioning that during the period of this investigation the epidemic in China was controlled. However, at the same time, the rest of the world was in the midst of a rapid outbreak, people were living in dire conditions, and the global hazards caused by the COVID-19 pandemic were far from over. Therefore, this survey will have a good reference value for countries that adopt ‘limiting to go out’ measures and also provide a basis for the active intervention of residents’ quality of life in similar public health emergencies in the future.In order to cope with the outbreak of the COVID-19 epidemic, this survey needed to evaluate the lifestyle of the population in a short period of time. So, a rapid epidemiological assessment was adopted. Due to the “stay at home” regulation of the Chinese people, the on-site survey could not be carried out and an online questionnaire could only be chosen. Accordingly, a cross-sectional sample of 1040 Chinese residents aged 16–70 years was recruited online via our research group from 29 March to 5 April. The gender, age, urban and rural areas, family income, educational level, and so on of the collected population were taken into account. These people were all voluntary participants, which greatly reduced the non-response bias that often exists in the on-site survey. However, as our research group is located in the Jiangsu Province, according to the survey results, residents in the Jiangsu region accounted for the largest proportion (68.46%), followed by nearby places such as Shanghai City (8.17%) and Zhejiang Province (4.23%), and the remaining 26 provinces together accounted for 19.14%. Therefore, this survey may be not a good representation of all Chinese residents. However, under such urgent conditions at that time, it more or less reflected the lifestyle of the residents in the most economically developed and ideologically active regions of China for the first time.All the participants were informed of the purpose and procedures of the study and participated voluntarily and anonymously. This study was approved by the ethical review committee of Nantong University (Ethics number: 202062).A very popular professional questionnaire platform, named ‘Wen Juan Xing’ in Chinese or ‘Sojump’ in English, was used to develop the questionnaire survey system. Through several simulation tests, the content of the questionnaire was continuously improved. Key content areas were developed by applying the socio-ecological model, including responders’ demographics, current diet, physical activity, and sleep behaviors, as well as changes before and during the outbreak. It should be noted that this study was a cross-sectional survey that collected the data at the time of the outbreak, while the data before the COVID-19 outbreak were recorded by the responders through their recollections. For those subjective qualitative questions, it was reported using a five-point Likert type scale, ranging from “a lot less” (score = 5) to “no change” (score = 3) to “a lot more” (score = 1). This self-designed questionnaire consisted of four parts, as follows.Gender, date of birth, living in the urban or rural areas during the epidemic, family per capita monthly income, resident population, educational background, body height, and body weight were included.(1)Change of dietary intake before and during the epidemic, including 10 categories: total intake, staple foods, vegetables, dairies, legumes or products, livestock/poultry meats, fished or fishery products, eggs, fruits, snacks/beverages;(2)Six psychological emotions on dietary behavior before and during the epidemic: dysphoria, anxiety, sadness, nervous, loneliness, depression; and(3)Dietary restriction, overeating, and storing food behavior during the epidemic.(1)Physical activity time, sedentary time, outdoor exercise time, and average daily steps before and during the epidemic;(2)Time spent using mobile phones and computers and doing housework during the epidemic; and(3)Weekly frequency of vigorous exercise and moderate exercise, as well as going out and walking during the epidemic.(1)The time to go to bed, fall asleep, and get up; the length of sleep; the quality of sleep before and during the epidemic;(2)Factors affecting sleep during the epidemic: disturbance in respiration, cough or snore, feeling cold or hot, having a nightmare; pain or ache; and(3)Feeling sleepy and undynamic during the epidemic.n = 100) and correlation coefficient range = 0.76–0.98 (strong reliability).Moreover, for every certain number of questions in the questionnaire, a quality control question was inserted to ask the respondents to choose the specified answer, to find the respondents who give a random answer. Test-retest (one week) reliability was assessed via bivariate Pearson correlation  before and after the COVID-19 outbreak. Analysis of variance (ANOVA) was used for comparison of weight gain between groups . The confounders  were adjusted for by analysis of covariance (ANCOVA). The count data were expressed by frequency (n) and percentage (%), and Pearson’s χ2 test was used for comparison between groups. The linear regression equation between the total score of the three main factors  and weight gain was calculated. Multiple linear regression modeling was used to analyze the interactions between the changes in total food intake, changes in physical activity, and the pre-outbreak overweight/obesity effect on weight gain. A p-value < 0.05 was considered statistically significant.In this study, the quantitative data were expressed as mean value ± standard deviation (mean ± SD). A paired Excluding the invalid and illogical data, the final number of respondents was 889. Among them, 347 (39%) were males and 542 (61%) were females. The ages ranged from 16–70 years old and the average was 31.8 ± 11.4 years. For residence type, 661 (74.4%) people lived in urban areas and 228 (25.6%) people lived in rural areas. During the COVID-19 outbreak, 607 (68.3%) people lived in Jiangsu province, 71 (8.0%) people lived in Shanghai city, 36 (4.0%) people lived in Zhejiang province, and 175 (19.7%) people lived in the other 31 provinces except Hainan, Ningxia, Hong Kong, Macao, and Taiwan. The education background was 78 (8.8%) completed junior high school, 116 (13.0%) completed senior high school, 618 (69.5%) completed bachelor’s degree, 57 (6.4%) completed master’s degree, and 20 (2.2%) completed doctoral degree. Per capita monthly household income was 211 (23.7%) people with less than 3000 yuan ($462), 252 (28.3%) people with 3000–6000 yuan ($462~924), 211 (23.7%) people with 6000–10,000 yuan ($924~1540), 143 (16.1%) people with 10,000–20,000 yuan ($1540~3080), and 72 (8.1%) people with more than 20,000 yuan ($3080). Permanent resident population was 1 population, 15 (1.7%); 2 population, 73 (8.2%); 3 population, 355 (39.9%); 4 population, 228 (25.6%); 5 population, 155 (17.4%); 6 population, 40 (4.5%); and 6 population and above, 23 (2.6%).Changes in food intake, physical activity, sleep duration, and sleep quality were rated on a scale of five compared to before the outbreak. The detailed results are shown in p = 0.074), with an average weight gain of 0.5 ± 2.8 kg. Firstly, through the one-way analysis of variance (ANOVA), it was found that changes in total food intake (p = 0.000) and physical activity (p = 0.020), as well as whether overweight/obesity before the COVID-19 outbreak (p = 0.004), had a significant effect on weight gain due to the “stay at home” lifestyle. After further adjusting for potential confounding factors by using the analysis of covariance (ANCOVA), the above three variables still had significant effects on weight gain, as shown in The proportion of respondents who gained weight (>1 kg) during the COVID-19 outbreak was 30.6% , age (p = 0.302), urban or rural residents (p = 0.312), per capita monthly household income (p = 0.408), permanent resident population (p = 0.384), and educational background (p = 0.390), as well as changes in sleep duration (p = 0.416) and sleep quality (p = 0.062) had no significant effect on weight gain. Other demographic indicators including gender  on weight gain, each individual was assigned a score, and then the sum was added to calculate the total score, so that the linear regression equation between the total score of the three main factors and weight gain was calculated .(1)p-interaction < 0.001)  A.(2)p-interaction = 0.001)  B.(3)p-interaction < 0.001)  C.Furthermore, the interaction analysis of the three main factors that significantly affect weight gain during the “stay-at-home” lifestyle was carried out, and it was found that:Lastly, all variables correlated with weight gain during the COVID-19 outbreak are listed in The purpose of this study was to assess the immediate changes in food intake, physical activity, and sleep quality in Chinese residents during the initial period of the COVID-19 outbreak. We found that Chinese residents had more food intake, were less active, were more sedentary, and gained weight during the initial period of the COVID-19 outbreak compared with before the restrictions. These changes did not differ in demographic indicators such as gender, age, urban or rural areas, household income, educational background, and so on. The largest changes reported were the increased intake of snacks/beverages and decreased outdoor activity. These findings confirm speculations that pandemic-related confinements were unfavorably related to lifestyle. This observation had triangulated support from quantitative  and qualitative  evidence.The COVID-19 outbreak is a classic public health emergency. A review reported negative psychological effects after isolation, including post-traumatic stress symptoms, confusion, and anger . AccordiSimilarly, in this study, we found that about 40% of the respondents had different degrees of increase in total food intake, and the increase in food intake was probably associated with psychological factors. The rapid outbreak of COVID-19, its high infectivity, and long epidemic period, together with the various lockdown measures adopted, constitute a stressor for the general public, making some especially vulnerable groups more anxious and depressed than before the outbreak. Either acute mild stress or prolonged chronic stress can influence our appetite, including our desire to eat and the types of food we are likely to choose . StudiesIn terms of energy consumption, due to the suspension of work and production, as well as the closure of fitness venues and public playgrounds, people’s exercise during the epidemic period was significantly reduced, replaced by more screen time. This survey found that more than half of the respondents had reduced physical activity during the COVID-19 outbreak compared with before (with a “significant decrease” accounting for 31.5%), including decreased outdoor activity, decreased step number, and increased sedentary time, which were also significantly associated with weight gain. Studies have shown that the abrupt interruption of physical exercise and prolonged inactivity may promote many adverse health changes, including the development of insulin resistance, muscle atrophy, bone loss, decreased aerobic capacity, increased blood pressure and heart frequency, fatty liver disease, nonalcoholic steatohepatitis, and dyslipidemia, as well as a higher risk of collapsing upon resuming exercise . TherefoContrary to previous studies that people with overweight and obesity were more likely to gain weight ,35, thisMoreover, the three factors most significantly associated with weight gain, namely, increased food intake, reduced physical activity, and non-overweight and obesity before the outbreak were analyzed through interaction analysis and multiple regression analysis. It was found that when these three main factors were combined, respondents had a higher risk of weight gain. Additionally, while this study found a slight increase in “sleep time” and “sleep quality”, there was no correlation with weight gain. But another domestic study found that after the outbreak of COVID-19, residents’ sleep quality declined, with an obvious sleep disorder . Certainly, in addition to the weight gain, there were also some harmful health problems that were not involved in this study, such as behavioral addiction disorders, inadequate sunshine exposure, and social isolation . In partThis study provided data on lifestyle changes during the COVID-19 lockdown about Chinese residents for the first time. There was an increase in total food intake by 39% of respondents, especially in snacks and drinks. The major psychological factors leading to the increase in food intake were loneliness and anxiety. There was a decrease in physical activity by 54.9% of respondents, especially in outdoor activities. The rate of weight gain in the total population was 30.6% and the average weight gain was 0.5 ± 2.8 kg. The main factors contributing to weight gain were increased food intake and reduced physical activity, and normal-weight people were more likely to gain weight than overweight or obese people during the “stay at home” lifestyle caused by the COVID-19 outbreak. Furthermore, the three main factors associated with weight gain interact with each other and can have a synergistic effect on weight gain.