Fat metabolism is associated with prediabetes and type 2 diabetes mellitus (T2DM). The aim of this study was to evaluate the detailed correlation of diabetes status with adiposity among adults.Briefly, 28,429 adults aged ≥18 years from both sexes in the National Health and Nutrition Examination Survey (NHANES) 1999–2018 were included in this study. Multivariable linear regression models were used to examine associations of prediabetes and diabetes status, disease duration of T2DM, serum glucose, glycohemoglobin (HbA1c) with total percent fat (TPF), and fat mass distribution.After adjusting for sociodemographic covariates, health behaviors, hypertension, hypercholesterolemia, there were direct associations of prediabetes and T2DM status with TPF, trunk fat mass, android fat mass, gynoid fat mass and android to gynoid ratio compared with non-diabetes. But the fat mass decreased with the increase of the disease duration in patients with T2DM. Besides, when stratifying by diabetes status, we found direct associations of serum glucose and HbA1c with TPF, trunk fat mass, android fat mass, gynoid fat mass, and android to gynoid ratio in non-diabetic and prediabetic participants. But in patients with T2DM, inverse associations of serum glucose and HbA1c with fat mass were observed.This study indicated that adults with prediabetes and T2DM had significantly higher TPF, trunk fat mass, android fat mass, gynoid fat mass, and android to gynoid ratio compared with those without diabetes. Moreover, fat mass decreased as the disease duration increased in patients with T2DM. The associations of serum glucose and HbA1c with TPF and fat mass distribution in patients with T2DM were opposite to the relationships observed in non-diabetic and prediabetic participants. Prediabetes, with blood glucose concentrations higher than normal, but lower than the threshold of diabetes mellitus (DM), is a high-risk state of DM development . The NatPatients with prediabetes or T2DM need to monitor glycohemoglobin (HbA1c) and blood glucose concentration regularly to prevent the development and progression of microvascular and macrovascular complications . CompareNational Health and Nutrition Examination Survey (NHANES) is a continuous surveillance survey conducted by the Centers for Disease Control and Prevention (CDC) and the National Center for Health Statistics (NCHS) to assess the health and nutritional status of the US population. Data obtained from NHANES can be freely available to researchers worldwide. In our study, we pooled data from 10 2-year cycles of NHANES 1999–2018.A total of 101,316 participants were enrolled from the NHANES 1999–2018 database. Among the 59,204 adults aged ≥18 years old, we excluded 28628 participants with incomplete TPF data, 1,763 participants with incomplete serum glucose data, 36 participants with incomplete HbA1c data, and 18 participants who had unclear self-report DM status. To minimize the number of participants with T1DM, 330 participants with age of DM onset before the age of 30 were also excluded. Finally, 28,429 participants were analyzed after applying these exclusion criteria .The National Center for Health Statistics Research Ethics Review Board reviewed and approved NHANES, and all participants signed written consents prior to participating in each year's survey. De-identified data are accessible online.The outcomes of our study were TPF, trunk fat mass, android fat mass, gynoid fat mass and android to gynoid ratio, which were measured by well-trained technicians using dual energy X-ray absorptiometry (DXA) QDR-4500 Hologic Scanner  of the whole body. TPF was calculated by the ratio of total fat mass to total fat and lean mass and multiplied by 100 to produce a percentage. The regions of fat distribution were defined by the Hologic APEX software. Specifically, the trunk fat mass was defined as total fat mass minus fat in limbs and head. The android area was defined as the lower trunk area bounded by two lines: the pelvic horizontal cut line on its lower side, and a line automatically placed above the pelvic line. The upper gynoid line was placed 1.5 times of the height of android region below the pelvic line and the lower gynoid line was placed such that the distance between the two gynoid lines was twice the height of the android region. DXA scans were not performed for participants with a self-reported use of radiographic contrast material (barium) in the last seven days before scans, weight above 450 pounds or height above 192.5 cm.Exposures were prediabetes and T2DM status, disease duration in those with T2DM, serum glucose and HbA1c. The T2DM status was defined as self-reported physician-diagnosed diabetes (ever told by a doctor that they had DM) or using measures of HbA1c ≥6.5% in those without a self-reported diagnosis, based on the guideline from the American Diabetes Association . The prehttp://www.cdc.gov/nchs/nhanes/).Sociodemographic variables mainly included age, sex, race and ratio of family income to poverty threshold. Current insulin use and glucose-lowering medication intake were assessed by questionnaires. Health behaviors variables included smoking status (whether smoked at least 100 cigarettes in life) and vigorous work activity. Health-related variables were hypertension (ever told by a doctor that you have high blood pressure), hypercholesterolemia (ever told by a doctor that you have high cholesterol), BMI and waist circumference . The detailed description of other variables such as total cholesterol, HDL cholesterol, triglyceride, LDL cholesterol as well as blood urea nitrogen, uric acid, creatinine can all be found on the NHANES website (http://www.Rproject.org) and Empower Stats (http://www.empowerstats.com), p-values <0.05 were considered to indicate statistical significance. The associations between diabetes status, duration of T2DM, serum glucose, HbA1c and TPF or fat distribution were evaluated by multivariable linear regression models. The model was adjusted for age, sex, race, health behaviors , hypertension, and hypercholesterolemia. Subgroup analyses stratified by sex and diabetes status were also performed.The NHANES sample weights were used as recommended by the NCHS. All analyses were performed with package R (2) (The weighted distributions of the characteristics according to diabetes status were shown in 2) , 11. Comp-values <0.0001. That is to say, after controlling for potential confounding factors, compared with those without DM, the TPF, trunk fat mass, android fat mass, gynoid fat mass and android to gynoid ratio for prediabetes were 2.12%, 3032.89 g, 609.47 g, 595.10 g, 0.06 higher, and for T2DM were 2.49%, 4937.83 g, 944.87 g, 582.69 g, 0.08 higher. In the subgroup analysis stratified by sex, this direct association existed in both males and females after adjusting for confounders. These results are presented in We found the direct associations of prediabetes and T2DM status with TPF and fat distribution compared with non-diabetes in fully adjusted models . The results were all statistically significant with CI: −0.16~−0.05; serum glucose and trunk fat mass: β = −79.49, 95% CI: −147.53~−11.46; HbA1c and TPF: β = −0.13, 95% CI: −0.24~−0.01). Other associations were of no statistical significance. The results are shown in After adjusting for sociodemographic covariates, health behaviors, hypertension, and hypercholesterolemia, we found direct associations of serum glucose and HbA1c with TPF, trunk fat mass, android fat mass, gynoid fat mass, and android to gynoid ratio. And when stratifying by diabetes status, these direct associations still existed in participants without diabetes and with prediabetes. But in patients with T2DM, the correlation of serum glucose with TPF and trunk fat mass, HbA1c with TPF turned to be inverse , which could be explained as each unit (1 year) increase in the duration of T2DM, TPF was decreased by 0.04% and gynoid fat mass was decreased by 18.41 g. When stratified by sex, the inverse associations of disease duration with TPF and gynoid fat mass were still statistically significant in females, but not in males. Besides, the inverse associations of disease duration with trunk fat mass, android fat mass and android to gynoid ratio were of no statistical significance either. The results are shown in The results of our study showed that individuals with prediabetes and T2DM had significantly higher TPF, trunk fat mass, android fat mass, gynoid fat mass, and android to gynoid ratio compared with those without DM. The fat mass decreased as the disease duration increased in patients with T2DM. Moreover, in participants without DM and with prediabetes, serum glucose and HbA1c were directly associated with TPF, trunk fat mass, android fat mass, gynoid fat mass, and android to gynoid ratio, while the inverse associations were observed in those with T2DM.Prediabetes and T2DM are associated with the increased insulin resistance in target organs  and pancreatic β-cell dysfunction, with nearly 50% cell loss at the diagnosis of T2DM , 12. AltThe present study also showed that TPF, trunk fat mass, android fat mass, and gynoid fat mass decreased as the disease duration of T2DM increased, although it was of no significance for trunk fat mass and android fat mass. This may be because once an individual was diagnosed with T2DM, the use of anti-diabetic drugs, the intervention of diet, and exercise may cause weight loss and decrease in fat mass, and the elimination of blood sugar through urination at an extremely high sugar level may also make some contributions . A largeThe blood glucose and HbA1c are considered as measures of DM control and parameters in relation to the risk of complications for decades. Of the two, HbA1c is more stable and convenient because of its absence of fasting . In thisThe main strengths of this study are the availability of a large, nationally representative population of US adults with data of fat mass and diabetes status from NHANES. Additionally, the availability of body composition measures by DXA offers additional information compared to the traditional measures of adiposity. More importantly, a large enough sample size allowed us to make the subgroup based on diabetes status and showed the distinct but neglected pattern of serum glucose and HbA1c with TPF and fat distribution that had never been reported in the previous studies.This study also has several limitations. First, it is a cross-sectional study, which limits the inference of a causal correlation between serum glucose, HbA1c and TPF, fat distribution among adults. So, further basic mechanism research and large sample prospective study are still needed to identify the exact mechanism between them. Second, some NHANES participants were not eligible for a DXA scan because of excessive weight, height, or other reasons, so the estimates in this study might not fully represent the TPF and fat distribution in the general population. Third, there remains the possibility of bias caused by other potential confounding factors that we did not adjust for. Furthermore, some associations of disease duration, serum glucose, and HbA1c with adiposity in patients with T2DM were of no clinical or statistical significance, which may mainly because the number of patients with T2DM was small, so study on a larger sample of patients with T2DM is eagerly needed. Besides, TPF defined by DXA may have several limitations, so the body fat mass and distribution in prediabetes and T2DM requires in-depth research in multiple areas such as diagnostic criteria for obesity in T2DM patients using TPF measured by DXA, alteration in fat mass since prediabetes or T2DM is diagnosed and its detailed mechanisms.Our study indicated that adults with prediabetes and T2DM had significantly higher TPF, trunk fat mass, android fat mass, gynoid fat mass, and android to gynoid ratio compared with those without DM, and the fat mass decreased as the disease duration of T2DM increased. We also found inverse associations between serum glucose, HbA1c, and fat mass in patients with T2DM and direct association in those with prediabetes and without DM participants, which may give us a hint that just the measurements of serum glucose, HbA1c, or fat mass and distribution may be insufficient to monitor the development and treatment effect of T2DM.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The Ethics Review Board of the National Center for Health Statistics approved all NHANES protocols.JS and ZL contributed to data collection, statistical analysis, and writing and revising of the manuscript. ZZh and ZZe contributed to statistical analysis. WK supervised the study and contributed to polishing and reviewing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the CSCO-ROCHE Research Fund No. Y-2019 Roche-015, Beijing Xisike Clinical Oncology Research Foundation Y-HS2019-43, Wu Jieping Medical Foundation No. 320. 6750.19020 and 320.6750.2020-08-32, and CAMS Innovation Fund for Medical Sciences No. 2020-I2M-C&T-B-027.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A table-top microdevice was introduced in this work to produce ultrasmall particles for drug delivery via inhalation. The design and operation are similar to that of spray-drying equipment used in industry, but the device itself is much smaller and more portable in size, simpler to operate and more economical. More importantly, the device enables more accurate control over particle size. Using Flavopiridol, an anti-inflammation medication, formulations have been developed to produce inhalable particles for pulmonary delivery. A solution containing the desired components forms droplets by passing through an array of micro-apertures that vibrate via a piezo-electrical driver. High-purity nitrogen gas was introduced and flew through the designed path, which included the funnel collection and cyclone chamber, and finally was pumped away. The gas carried and dried the micronized liquid droplets along the pathway, leading to the precipitation of dry solid microparticles. The formation of the cyclone was essential to assure the sufficient travel path length of the liquid droplets to allow drying. Synthesis parameters were optimized to produce microparticles, whose morphology, size, physio-chemical properties, and release profiles met the criteria for inhalation. Bioactivity assays have revealed a high degree of anti-inflammation. The above-mentioned approach enabled the production of inhalable particles in research laboratories in general, using the simple table-top microdevice. The microparticles enable the inhalable delivery of anti-inflammation medicine to the lungs, thus providing treatment for diseases such as pulmonary fibrosis and COVID-19. Inflammation in the lungs is a principal cause of diseases, including lung fibrosis ,2, lung Inhalable particles are typically produced using a spray-drying technique, involving the rapid drying of liquid formulations into dry powders that are immediately ready for use ,29,33. W2), Tween-20, and Ethanol 100% were purchased from Sigma Aldrich . Phosphate-buffered saline (PBS) (1X) was purchased from Mediatech . Gaseous nitrogen was purchased from Praxair . Poly (tetrafluoroethylene) (PTFE) tubing was purchased from Cole-Parmer , and 96 well plates, 10- and 20-mL glass scintillation vials, and 1.00 mm ID glass capillary tubes were purchased from Fisher Scientific . The variable area flowmeter was purchased from Dwyer Instruments Inc , and Dulbecco’s modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), and 2X Lysis reagent were purchased from Invitrogen . Human embryonic kidney 293 cells (HEK-293) were purchased from ATCC . Nuclear factor kappa B .1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Avanti Polar Lipids . Flavopiridol was purchased from Cayman Chemical Company . L-leucine, L-isoleucine, D-(+)-Glucose, Calcium chloride (CaClv/v) ethanol solution. The dispersion was sonicated for 15 min until complete dissolution of the particles was complete. The dissolved solution was loaded in a 1 mL syringe and filtered into a quartz cuvette using a 0.2 v/v) ethanol/water. Then, the concentration of Flavopiridol (c) was calculated using Beer’s law. The encapsulated mass of Flavopiridol .The encapsulation efficiency (EE) of Flavopiridol was quantified using Equation (2) by dividing the LC with the initial loading capacity in the organic phase, also known as the desired loading capacity of Flavopiridol, in this case, The microparticle geometry and size were determined using a field emission scanning electron microscopy (SEM) . Particles were immobilized to a double-sided carbon tape  supported by an Al stub . After coating the particles with 10 nm of gold using a sputter coater , the samples were transported to the SEM vacuum chamber. The typical acceleration voltage and emission current used in this work were 2 kV and 10 The aerodynamic diameter da=dv/v)% Tween-20 was added to establish sink conditions. The particle dispersion was heated to 37 In vitro release experiments were carried out to determine Flavopiridol release from the inhalable particles utilizing demonstrated release protocols ,35. BrieThe in vitro release kinetics were quantified by fitting the measured profile using a 2-term model established previously ,37, as iThe first term represented the process of “burst release”, with solution . The 2nd release ,40,41. T2 to represent a near physiological environment ,43. For each exposure condition, six 1.5 mL Eppendorf tubes were used, each filled with 1–2 mg of freshly prepared particles under ambient laboratory environment, 24 °C with relative humidity (RH) typically read at 40%. After exposure to the environment to a designated time, a vial was taken out, to which a 0.5 mL of 70% ethanol was added and then sonicated for 15 min until complete dissolution was achieved. The solution was filtered through via a 0.2 These particles were subject to exposure to three different environments. The first environment was set to 4 geration ,43. The Attenuated Total Reflectance—Fourier Transform Infrared Spectroscopy (ATR-FTIR)  was usedThe anti-inflammation activity of Flavopiridol released from inhalable particles was determined by a luciferase reporter assay as described previously ,45. Brie2O)1/2 min/L. The EPM is calculated below, as seen in Equation (6):The aerodynamic performance studies followed the pharmaceutical industry’s good practice and were carried out by iPharma Labs, Inc . The emitted powder mass (EPM) was measured from the emitted aerosol dose from a RS01 dry powder inhaler (DPI). Briefly, 5 mg of inhalable particles was filled into size 3 clear capsules and loaded (2O)1/2 min/L.The inhalable particles were delivered from an RS01 DPI, and the aerodynamic performance was tested using a next-generation impactor (NGI)  using a modified NGI gravimetric cups , fitted with 55 mm glass fiber filters  as impaction surfaces. The NGI set up consisted of seven stages and a micro-orifice collector (MOC). An internal filter for the MOC stage ) with a 76 mm glass fiber filter  was used in all test runs. For each test run, two capsules  were filled with 5 mg of inhalable particles. Each capsule was loaded into the RS01 device, and an aerosolized mass was collected by the NGI at 60 L/min, which corresponds to 4 kPa drop for RS01 having a flow resistance of 0.10  using Karl Fischer titration on a coulometric Nittoseiko Analytech Moisture Meter CA-310 with a frit-less cathode and Vaporizer VA-300 . In a glove box, 60 mg of inhalable particles was loaded into a capped 4 mL glass vial  and then analyzed at 100 ® CAD software . A 50 mL plastic BD syringe  served as a holder for the feed solution, whose back was pushed by a syringe pump . The solution exited to a 23 AWG PTFE tubing tightly connected to a 22 AWG blunt needle . 2 cm below the needle was a stainless steel (SS) plate with an array of orifices . The two piezo rings were fixed atop and at the bottom of the SS plate, respectively, serving as the actuator. The piezo actuator was controlled by our home-constructed piezo driver at 10–150 kHz and 0–30 V amplitude, and 50 cm below the plate, a custom PTFE funnel (D = 21 cm opening) served as the initial liquid droplet collector, with the narrow exit connect to our custom cyclone chamber . The cyclone separator adopted a cylinder-on-cone design with a tangential inlet and was manufactured in PTFE. The exit was threaded to a glass container to collect solid particles. The vacuum suction was from the top center of the chamber via a silicone tubing connecter to a mechanical pump .The home constructed table-top microdevice (TTMD) was illustrated schematically in 2 area of the stainless steel (SS) plate with orifice array. The vibration of the plate interrupted the liquid flow into a spray of small droplets. By varying the aperture size and/or vibrational frequency, microfluidic devices enable the tuning of droplet size [During operation, the feed solution was pushed into the PTFE tubing through the syringe pump at a designated flow rate. The solution exited through the blunt needle showering ~1 cmlet size . Using oIn this TTMD, the initial volume of liquid is up to 50 mL per batch, sufficient to produce up to 250 mg of particles per hour. A typical dosage of inhalable particles is around 5 mg for humans ; thus, ew/w) based on the definition of LC shown in Equation (7).As the initial estimation of Flavopiridol concentration in the solution before activating microfluidic synthesis, we examined the practical factors pertaining to human inhalation. Enroute to the lung for being uptaken, a portion of inhalable particles would reach the epithelial lung lining fluid (LLF), where gradually, Flavopiridol would get released and become soluble and thus bioavailable in LLF for human uptake . A typicThe additional consideration of LC includes the loss of the drug before reaching LLF. It was reported that nearly 25% of the encapsulated drug could reach the intended destiny in cases of pulmonary delivery using a DPI ; therefoBesides drug dosimetry, further considerations include inhalability and non-aggregation for the resulting solid microparticles ,52. The μm ,53, fullμm , and higμm ,54.The next consideration is selecting solvents suitable for our TTMD; followed by selecting and varying the excipients and concentration of Flavopiridol to reach the desired particle morphology, and dispersion; and, finally, varying experimental parameters (see above) to reach the designed size for inhalation.For measurements of particle size and for visualizing particle morphology, our field-emission SEM was utilized. Unlike standard and conventional SEM imaging of drug delivery particles ,44, our Briefly, <1 mg of powder formulations was inserted into a 1.00 mm ID glass capillary tube and mounted vertically via three-fingered clamps. The end of the capillary tube was custom-fitted with a syringe-needle port , which was inserted into a 21 AWG PTFE tubing. The end of the PTFE tubing was directly fitted to a variable area flowmeter  attached to nitrogen gas tank. The flow rate of nitrogen was set at rates comparable to the nominal human breathing at sea level, 6–16 L/min . The floTo assure complete solvent evaporation using our TPMD, a mixed solvent of ethanol:water with volume ratio ranging from 50:50 to 90:10 was tested. The purpose was to assure the solubility of Flavopiridol and of all excipients in a single phase in the beginning to attain dry particles at the collector upon completion of synthesis. The optimized ratio for this work was determined to be ethanol:water = 70:30.2 to phospholipid formulations resulted in improved environmental robustness [2 resulted in heterogeneity in shape with a flattened heterogenous morphology; therefore, it was not beneficial for this synthesis. The addition of sugars, such as glucose, has been utilized in prior synthesis as an enhancer for bioavailability during pulmonary delivery [w:w.In the design of inhalable particles, excipients or non-active components represent major components of the particle, thus dictating the physio-chemical properties and inhalability of the particles ,32. Manybustness . As seendelivery ,64. In ow/v) [Assuming all components in the solution are equal to the final composition of the particles, the final formulation would be L-isoleucine:DPPC:Flavopiridol = 89.8:10.0:0.19. The typical total concentration used in spray drying was in the range of 0.1–0.5% (w/v) . In thisFinally, with the formulations at hand, we varied the TTMD operation parameters systematically until the geometrical diameter of the particles was around rphology A and higw/w), which is lower than other reported inhalable particles [Under the specified conditions  to produarticles  and comparticles .The size, morphology, and dispersity of the optimized particles were determined via SEM and are shown in delivery . These ITo verify that the active component, Flavopiridol, retained its structural integrity and chemical functionality in our TTMD synthesis, we measured the UV-Vis spectra for Flavopiridol as received, and that from fully dissolved ILDF particles. The similarity of the two spectra, shown in The Flavopiridol concentration in ILDF can be quantified from the spectra at −1 [To check and verify the chemical integrity of other components in our ILDF microparticles, ATR-FTIR spectroscopy was acquired. As shown in ed star) , and theck star) . The pretar) [−1  (blue stn = 3) is shown in The testing of the aerodynamic performance of our ILDF particles followed the industry’s standard of good practice and was carried out through measurements of EPM, MMAD, FPF, and GSD using an NGI from the emitted aerosol dose from a RS01 DPI. The EPM was measured from the emitted aerosol dose as the average of five capsules with nominal filled mass of 5 mg. The EPM was determined to be 96.5 products . The meaThe particle size fractionation distribution is observed in ± 0.6 μm  calculatdelivery ,29,33. Tribution . The FPFIn vitro release experiments were carried out for the Flavopiridol loaded ILDF particles, utilizing the protocols described in The quantification of release kinetics follows the known kinetics model, described in  studies . This ra studies ,71.2, to represent a near physiological environment [The ILDF particles , row 2 wgeration ,43. The ironment ,43.At 4 wn in °C , middle,To test the physical stability and structural integrity of the produced ILDF particles, the particles subject to the harshest exposure conditions among the three, i.e., 40% RH, 24 These results are very encouraging, well positioning our products as candidates for further investigation, including in vivo and preclinical testing.The biological activity of Flavopiridol in the ILDF particles was assessed using a luciferase assay ,45. The In comparison, as a positive control, cells were treated with 300 nM of soluble Flavopiridol (blue bar). It is clear that the luciferase activity became oppressed dramatically, due to the presence of Flavopiridol, limiting the luciferase expression. The performance of the Flavopiridol in the ILDF particles (red bar) matched the positive control with little statistical difference. These measurements suggest that the biological activity of Flavopiridol released from the ILDF particles was retained. These tests paved the way for further investigations of pulmonary delivery in vivo in the near future.A table-top microdevice was designed and constructed to produce ultrasmall particles for drug delivery via inhalation. The design and operation were very similar to that of a spray-drying equipment used in industry, but the device itself is much smaller and portable in size, more accurate in particle size control, and simpler to operate and economical. Using Flavopiridol as an anti-inflammation medication, formulations of the initial liquid were developed. The liquid formulation droplets passed through the micro-aperture arrays of the device that was shaken via a piezo-electrical driver. High-purity (99.99%) nitrogen gas was introduced and flew through the designed path, including the funnel collection and the cyclone chamber, and finally was pumped away. The gas carried and dried the micronized liquid droplets along the pathway, leading to the precipitation of dry solid microparticles. The formation of cyclone was essential to assure the sufficient travel path length of the liquid droplets to allow for the drying and formation of solid particles. Synthesis parameters have been optimized to produce microparticles, whose morphology, size, physio-chemical properties, and release profile met the criteria for inhalation. The aerodynamic testing, including MMAD, FPF, and GSD determination, concluded that the ILDF particles revealed excellent aerodynamic performance. Bioactivity assays have revealed a high degree of anti-inflammation. The advantages are clearly demonstrated, including a high degree of consistency in particle size and composition and simplicity in device fabrication and usage. Thus, this approach enables the production of inhalable particles in research laboratories in general. The generality of the microdevice also allows for the production of a wide range of drugs beyond Flavopiridol, including Atuveciclib , LDC0000


CD27, IL7R, CXCL1, CCR7, IGLL5, and CD79A) were obtained by intersecting hub genes with DEGs, followed by verifying the diagnostic accuracy with the receiver operating characteristic curve (ROC). In addition, we constructed the least absolute shrinkage and selection operator (LASSO) regression model for hub gene cross-validation. Finally, we found that CD27 and IGLL5 were good diagnostic indicators of LOD, and CD27 may be the key gene of immune function change in LOD. In conclusion, our research shows that the changes in the immune function may be an important mechanism in the development of LOD, which can provide some guidance for the related research of LOD in the future.Late-onset major depressive disorder (LOD) increases the risk of disability and suicide in elderly patients. However, the complex pathological mechanism of LOD still remains unclear. We selected 10 LOD patients and 12 healthy control samples from the GSE76826 dataset for statistical analysis. Under the screening criteria, 811 differentially expressed genes (DEGs) were screened. We obtained a total of two most clinically significant modules through the weighted gene co-expression network analysis (WGCNA). Functional analysis of the genes in the most clinically significant modules was performed to explore the potential mechanism of LOD, followed by protein–protein interaction (PPI) analysis and hub gene identification in the core area of the PPI network. Furthermore, we identified immune infiltrating cells using the cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm between healthy subjects and LOD patients with the GSE98793 dataset. Next, six hub genes ( As a common mental disorder with high morbidity, major depressive disorder (MDD) is the main cause of severe disability in patients . The ageThe involvement of immune response in the process of MDD has been confirmed, such as immune cells and cytokines . Body fuIn recent years, bioinformatics methods have been widely used in different diseases, including LOD . A previAll datasets (GSE76826 and GSE98793) used in this study were downloaded from the Gene Expression Omnibus database. The GSE76826 dataset contains 12 healthy control individuals, 10 patients with LOD (onset age ≥50 years), and 10 patients with LOD syndromal remission. Also, 12 healthy control individuals and 10 patients with LOD were selected for the WGCNA. In addition, 12 healthy control individuals and 10 patients with LOD syndromal remission were selected for the cross-validation of hub genes. The GSE98793 dataset contains 64 patients with MDD, 64 healthy controls, and 64 patients with MDD and generalized anxiety disorder. From the GSE98793 dataset, we selected healthy controls and MDD patients without anxiety disorder for the immune infiltration analysis, whose age ≥50 years. We used the Perl language (version 5.32.1) to convert the probe ID extracted from the downloaded series matrix into a gene symbol. After that, R software (version 3.6.1) was used on the gene expression datasets for normalization and statistics . We follp-value < 0.05 were defined as DEGs. According to the screening criteria for DEGs, we used R software with the “limma” package to complete the screening of DEGs. A heatmap and volcano map were obtained to visualize the top 100 DEGs (50 upregulations and 50 downregulations) through the “pheatmap” package in R software.Compared with the healthy control individuals, the genes with fold changes < −1.4 or >1.4 and an adjusted p-value less than 0.05 were considered the most clinically significant modules for LOD. The gene information in the most clinically significant modules was selected.We used R software with the “WGCNA” package to identify and visualize the co-expressed modules of the genes in the GSE76826 dataset . The conp threshold <0.05.We performed functional enrichment analysis on genes in the first two most clinically significant modules, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The functional enrichment results were visualized through corresponding R packages. The criteria for functional enrichment were the To explore the difference in the proportion of immune infiltrating cells  in healthy control individuals and patients with LOD, we used the CIBERSORT algorithm to analyze selected samples from the GSE98793 dataset. The results of the immune infiltrating cells were visualized by R software with corresponding R packages.Genes in the first two most clinically significant modules were selected to construct a PPI network through the Search Tool for the Retrieval of Interacting Genes (STRING) . The setWe applied the ROC to verify the diagnostic accuracy of the six hub genes in LOD. The value of the area under curve (AUC) of the ROC represented the diagnostic accuracy. We used R software with the “pROC” package to perform ROC analysis and visualize the results. In the GSE76826 dataset, healthy control individuals and 10 patients with LOD were defined as the control group and case group, respectively. According to the respective real expression values of hub genes in the two groups, the ROC curves were drawn using the “pROC” package with R software. Meanwhile, the ROC curves were smoothed using the “smooth” function, and the AUCs of the ROC curves were calculated for each hub gene. AUC values >0.7000 were defined as accurate. For the cross-validation, we re-drew the ROC curve of each hub gene, according to the actual expression values of hub genes in healthy control individuals and patients with LOD syndrome remission to approach the real testing power.According to the expressions of hub genes, we used R software with the “glmnet” package to construct the LASSO regression model. The parameters of the LASSO regression model were set as follows: family = “binomial” and alpha = 1. Tenfold cross-validation was utilized to determine the optimal value of the penalty parameter λ. Next, the genes screened by the LASSO regression model and the genes verified by the ROC were intersected to obtain the final genes. We used “ggpubr” and “limma” packages to visualize the expression level of the final genes. Finally, the correlations between final genes and immune cells were analyzed by Spearman’s method.As shown in p-value < 0.05.), we applied R software with the “limma” package to identify the DEGs between healthy control individuals and patients with LOD. A total of 811 DEGs, which consisted of 383 upregulated genes and 428 downregulated genes, were obtained. As shown in According to the defined criteria (fold changes < −1.4 or >1.4 and adjusted 2 = 0.88), which meets the approximate scale-free topology criterion . The light cyan module had the second significant correlation with LOD (r = −0.56 and p = 0.006) (As shown in = 0.006) . Therefo= 0.006) .n = 1,025) and light cyan module (n = 296) were selected to perform GO and KEGG enrichment analyses. The results of the GO analysis were divided into the biological process (BP), cellular constituent (CC), and molecular function (MF). As shown in the GO results, in the blue module, the BP of LOD was mainly manifested in T-cell activation, regulation of cell−cell adhesion, and leukocyte cell−cell adhesion. The CC of LOD was mainly manifested in the external side of the plasma membrane, collagen-containing extracellular matrix, and the apical part of cells. The MF of LOD was mainly manifested in carbohydrate binding, immune receptor activity, and cytokine receptor activity. Meanwhile, the results of the KEGG analysis showed that the cytokine−cytokine receptor interaction, hematopoietic cell lineage, and complement and coagulation cascades were the main manifestations. Similarly, as shown in the GO results, in the light cyan module, the BP of LOD was mainly manifested in B-cell activation, the immune response-regulating signaling pathway, and immune response-regulating cell surface receptor signaling pathway. The CC of LOD was mainly manifested in the external side of the plasma membrane, MHC class II protein complex, and MHC protein complex. The MF of LOD was mainly manifested in immune receptor activity, MHC class II protein complex binding, and MHC protein complex binding. In the KEGG analysis of the light cyan module, the hematopoietic cell lineage, intestinal immune network for IgA production, and B-cell receptor signaling pathway were the main manifestations. All functional enrichment results are shown in Genes in the blue module (p = 0.003), regulatory T cells (Tregs) (p = 0.034), and neutrophils (p = 0.049), whereas the gamma delta T-cell (p = 0.003) fraction was relatively lower. All the results are shown in A previous study confirmed that immune function changes play an important role in MDD . HoweverCD8A, MMP9, PLG, IL7R, CD27, ITGB2, WNT10A, SERPINE1, CXCL1, and CCR7. Meanwhile, in the light cyan module, the top 10 hub genes were CD19, CD79A, CD79B, IGLL5, BLK, CD22, TNFRSF13C, CD40, IGJ, and PAX5. The hub genes were screened by intersecting DEGs and the 10 hub genes of the blue module and light cyan module. Finally, the six hub genes were CD27, IL7R, CXCL1, CCR7, IGLL5, and CD79A. In addition, we performed functional enrichment analysis on the six hub genes. As shown in the GO results of the hub genes  are shown in ub genes , the BP CD27, IL7R, CXCL1, CCR7, IGLL5, and CD79A) through ROC verification. As shown in CD27, IL7R, CXCL1, CCR7, IGLL5, and CD79A) were 0.809, 0.622, 0.644, 0.417, 0.801, and 0.737  were obtained by overlapping the top 10 hub genes in the two modules with DEGs. The functional enrichment results of six hub genes of LOD were mainly related to the immune function, indicating that the six hub genes may be crucial to the immune process of LOD. Meanwhile, we confirmed the immune infiltrating cells to LOD through the gene expression profiles of the GSE98793 dataset. Finally, we obtained the final genes (CD27 and IGLL5) through cross-validation of ROC and LASSO regression and found that CD27 was significantly related to the immune correlation in LOD.MDD is a highly prevalent psychiatric disorder . MDD is CD27, IL7R, CXCL1, CCR7, IGLL5, and CD79A) of LOD, which were obtained by intersecting the results of DEGs and the PPI network, were mainly related to the immune function. Taken together, these results indicate that the immune response may be crucial to the development of LOD, and six hub genes may be the key to these immune response mechanisms.It has long been confirmed that inflammation plays an important role in the process of MDD . The decCD27 may lead to anxiety or depression in patients with systemic lupus erythematosus by regulating T cells  in depression has been reported. In addition, there are few studies on IL7R, IGLL5, and CD79A in depression. ROC and LASSO helped us screen CD27 and IGLL5 as the accurate predictors of LOD. In addition, we found that CD27 may be the target of immune function changes in LOD. Our research shows that the changes in immune function may be an important mechanism in the development of LOD. However, there are several limitations to the present study. The detailed mechanism of the final hub genes found in LOD needs to be further studied. In addition, the dominant immune infiltrating cells in LOD need to be confirmed, which may interfere with the development of LOD.In summary, we explore the difference between healthy subjects and LOD patients by using the WGCNA and immune infiltration. We confirmed the six hub genes and the immune infiltrating cells in LOD. Among the six hub genes, the role of three genes (


Those significant improvements in skin symptoms were accompanied by a significantly enhanced water content (conductance) and a significant improvement of roughness (SESC) and smoothness (SESM) values measured using a Visioscan at 3 and 6 weeks. Those effects appeared concomitant with a significantly increased corneocyte size, a significantly down-regulated degree of thick abrasions, and a significant impairment of the corneocyte lipid envelope at 6 weeks. Thus, our clinical study suggests, for the first time, that topical application of the POLG nano-emulsion has the distinct potential to ameliorate atopic dry skin symptoms, particularly scaling and itchiness, in the skin of patients with AD. Those effects result from alleviation of the disrupted water-holding function probably due to the increased supply of lamellae structures into the stratum corneum despite the failure to improve barrier function.Because ceramide-like lipo-amino acid cholesteryl derivatives can exert a bound water-holding function due to their lamellae-forming properties, in this study, we determined if topical application of those derivatives to atopic dry skin would elicit an ameliorative effect on skin symptoms, at least on its water-holding function. In this clinical study, daily treatment with a nano-emulsion containing 10% phytosteryl/octyldodecyl lauroyl glutamate (POLG) significantly ( Atopic dermatitis (AD) is a recurrent type of skin dermatitis that has a high susceptibility to itching, irritants, and allergens, even in non-lesional skin, called atopic dry skin, with severe dry appearances (dryness) . AD is cThe combination of the disrupted barrier and water-holding functions in atopic dry skin is closely linked to the disease severity of AD ,16, whicThe most reasonable approach to improving these abnormalities in the SC is to topically apply natural skin ceramides at a high concentration to compensate for the ceramide deficiency in the SC of AD skin. Although we have already confirmed the distinct efficacy of natural ceramides by topical application on dry skin caused by treatments with solvents or surfactants ,18,19,20We have already reported that the atopic dry skin and water deficiency that occurs in the nonlesional skin of patients with AD , or thatp < 0.01) in those scores at 6 weeks (p < 0.001) and for 6 weeks (p < 0.0001) compared with week 0 (p < 0.05) and for 6 weeks (p < 0.0001) compared with week 0  both at 3 and at 6 weeks compared with week 0  and smoothness (SESM) A,B, alth2 h is substantially meaningless based on the evidence that less than 10 g/m2 h TEWL generally occurs in the skin of healthy subjects. These findings suggest that the POLG nano-emulsion has a considerable potential to increase the water content in the SC, whereas it has no significant ability to strengthen the barrier function, although the AD patients enrolled in this study had no deficiency in their barrier function compared with healthy controls [Treatment with the POLG nano-emulsion induced a significant amelioration in conductance values, with significant and marked increases at 3 and at 6 weeks  compared with week 0 A. On thecontrols . The 6 weeks of treatment with the POLG nano-emulsion induced a significant increase in corneocyte size A, which p < 0.001) improved several dry skin symptoms, including dryness/scaling, itchiness, and stimulus sensations in the non-lesional skin of patients with AD at 3 and at 6 weeks compared with week 0. Based on general clinical evaluation, the POLG nano-emulsion had 12.0, 8.0, 72.0, and 8.0% distribution after 3 weeks of treatment, and 12.0, 40.4, 44.0, and 4.0% distribution after 6 weeks of treatment, with marked improvement, moderate improvement, slight improvement, and no improvement, respectively. As we have already performed similar clinical studies [®(HIRU) cream (W/O type) had 0, 15.4, 80.8, and 3.9% distribution after 4 weeks of treatment. (2) In another comparative clinical study using the CER and HIRU creams [In this clinical study, the daily continuous use of the 10% POLG nano-emulsion significantly , reflecting non-barrier disrupted skin [In this study, improvements of atopic dry skin symptoms were confirmed by a dermatologist’s (KN) clinical scoring and by instrumental measures for water-holding functions (conductance), although improvements by instrumental measures for barrier functions (TEWL) were not detectable. It is likely that the lack of improvement in the barrier function may be attributable either to the healthy control levels of TEWL values  of the SC at 6 weeks is distinctly prominent in comparison with 175% (conductance) for the CER cream and 157% (conductance) for the HIRU lotion at 4 weeks , 169%  and smoothness (SESM) at 3/6 and 6 weeks, respectively. This improvement in scaling score also supports the fact that it appears concomitant with the significantly increased corneocyte size, the significantly down-regulated degree of thick abrasions, and the significantly impaired corneocyte lipid envelope. Further, the 199.7% improvement in water content of the SC at 6 weeks strongly suggests that the POLG nano-emulsion regulates the water-holding function in the SC, probably via the increased formation of lamellar structures into which water molecules are incorporated as non-evaporable bound water. The coordinated alleviations of all those factors strongly support the possibility that application of the POLG nano-emulsion to the skin may accelerate lamellae formation between the SC layers to enhance its water-holding function, resulting in a marked amelioration of desquamation, as well as a down-regulation of the turnover rate, which is responsible for regulating corneocyte size . During our previous clinical studies on AD dry skin using the CER cream or the HIRU lotion ,28, the This study has several limitations: first, we did not compare different vehicles and solvents to deliver the bioactives to the skin, and it is possible that other vehicles might be more or less efficient than the POLG nano-emulsion. Second, we do not have any preclinical data on the AD patients of this study concerning the disrupted water-holding function of their skin. Third, the SC water-holding capacity of the skin is also, in part, regulated by filaggrin in corneocytes, which can vary significantly in different subjects, and the filaggrin genotypes of the AD patients in this study were not assessed. Although clinical evaluations using the 10% POLG nano-emulsion on atopic dry skin should be conducted as a double-blind study with controls, which requires a non-treatment side, we did not set up a control side for the convenience of the enrolled patients with AD. Because this clinical test was carried out during the period from 10/15/2021 (autumn) to 12/17/2021 (winter) in Tokyo, Japan, it is well recognized that climate changes may not ameliorate the atopic dry skin.In conclusion, taken together, our clinical study suggests, for the first time, that the topical application of the POLG nano-emulsion has a distinct potential to ameliorate atopic dry skin symptoms, especially scaling and itchiness, in the skin of patients with AD by alleviating the disrupted water-holding function as a result of supplying lamellar structures into the SC despite the failure to improve barrier functions. This study also provides an insight into developing an effective skin care system for AD; it seems reasonable to assume that materials with the ability to form lamellar structures, even non-ceramide derivatives, are also suitable to ameliorate atopic skin symptoms in mild AD patients without severely disrupted barrier function. The nano-emulsion containing 10% phytosteryl/octyldodecyl lauroyl glutamate (POLG) was provided by ToutVert Co., Ltd. (Osaka Japan). The chemical structure of POLG is shown in This study (TV-2021-10-001) was performed from 15 October 2021, to 17 December 2021, at the Ebisu Skin Research Center, Inforward Co., Ltd., Tokyo, Japan. The nano-emulsion containing 10% POLG was applied twice a day for 6 weeks on the right or left forearm of 25 Japanese patients with AD.Diagnoses of AD were made according to the criteria of Hanifin and Rajka . The sevThis clinical test was registered under the number UMIN000037554. All procedures using human subjects were performed in accordance with the ethical standards of the Institutional and/or National Research Committee, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The present study adhered to the tenets of the Declaration of Helsinki, and was reviewed and approved by the Japanese Society of Aesthetic Dermatology Examination Ethics Committee (CrrC21-031). Formal informed consent was obtained from each subject before the study. Following screening by a dermatologist, 25 Japanese patients with AD with mild severity attended this evaluation.A trained dermatologist (KN) performed all clinical assessments. The evaluation parameters consisted of dryness, erythema, papules, and abrasions, and were scored on a 1–5 grading basis . Sensory attributes, such as stimulus sensations and itchiness, were assessed by querying each subject, and were scored on the same 1–5 grading basis.Clinical improvement was scored on a 0–3 grading basis  by comprehensively evaluating the clinical scoring. Thus, clinical improvement was evaluated according to the difference in dryness/scaling and itchiness score between before and after treatment as follows: when either score changed by one grade compared with before treatment, a slight improvement (1) was assigned. When both scores changed by one grade compared with before treatment, a moderate improvement (2) was assigned. When either or both scores changed by more than two grades compared with before treatment, a marked improvement (3) was assigned. Thus, it is likely that clinical improvement substantially reflects an efficacy rate for the amelioration of atopic dry skin symptoms.According to previously described methods ,38, skin2 area on the forearm of each individual, and the average of three values was used after removing the maximum and minimum values. TEWL, a parameter for the cutaneous barrier function of the SC, was measured using a Tewameter TMHex , and is expressed as g/m2·h. The measurements were repeated five times within a 2 cm2 area on the forearm of each individual, and the average was used. All measurements were performed in a climate-controlled room at 21 °C with an average humidity of 51.62% . To minimize regional anatomic differences, the ventral forearm was used as the measuring site whenever possible. Conductance, a parameter that reflects the water content of the skin surface, was measured using a SKICON-200EX-USB , and is expressed as µS. The measurements were repeated five times within a 2 cmCorneocytes were obtained by the tape-stripping method using cellophane tape . The tape was cut into a circle with a diameter of 6 mm that was tightly adhered to a glass slide to transfer corneocytes. Each glass slide was then immersed in xylene twice and then in ethanol to remove the cellophane by dissolving the adhesive agent. Corneocytes transferred to glass slides were subjected to the following examinations.Measurements of corneocyte size and the degree of thick abrasions were performed according to previously described methods . In brieMeasurements of the staining of lipid envelopes were performed according to previously described methods . In brieStatistical analyses were performed using GraphPad Prism 9 . For clinical data, we used ANOVA with the Dunn’s multiple comparisons test.p-values of 0.05 or less were considered statistically significant.For other data, normality was confirmed by the Shapiro–Wilk test. Data with more than three groups were analyzed by ANOVA, followed by Tukey’s multiple comparison test for parametric data, or by Dunn’s multiple comparison test for nonparametric data. Data with two groups were analyzed by a paired t-test for parametric data, or by the Wilcoxon matched-pairs signed rank test for non-parametric data.


The electrochemical polymerization of polypyrrole nanowires is carried out using potentiodynamic and galvanostatic methods in order to enhance the performance of the modified electrodes as capacitor devices. The electrochemical, spectroscopic, and morphological properties are determined through cyclic voltammetry, Raman spectroscopy and scanning electron microscopy, respectively, corroborating the presence of PPy-nw in dimensions of 30 nm in diameter. Characterization as a capacitor revealed that the nanowire structure enhances key parameters such as specific capacitance with 60 times greater value than bulk polymer modification, in addition to a significant increase in stability. In this way, it is verified that electrodes modified with polypyrrole nanowires obtained in situ by electrochemical methods constitute an excellent candidate for the development of capacitors. The mosacid [−1 ,13. Howeacid [−1 , both chacid [−1  and elecacid [−1 . The latacid [−1 ,18, whicacid [−1 , withoutp-doping process typically damages the three-dimensional structure of graphene, producing an internal resistance, which ultimately damages the electrode [p-doping/undoping charge [In this way, it has been reported that the electrodes constituted by CPs display better capacitance and processability, and greater cyclability—attributed to the improvement of the mechanical stability provided by the carbon-based compounds—used in the synthesis . For inslectrode . Therefog charge .In the present report, a method is proposed and tested to electrosynthesis PPy nanowires in situ from the electro-obtaining of mesoporous silicon oxide films reported by Walcarius et al. ,24,25. Fp-doping/undoping process is compared between nanostructured and bulk morphology, obtaining on electronic properties, with application in energy storage devices, such as capacitors.Based on this background, the PPy −1) cleaned and degreased in ultrasound for 5 min in acetone, 5 min in propanol, and finally, 5 min in milli-Q water. A large area spiral Pt wire is used as a counter electrode, and an Ag|AgCl electrode with potential adjusted to that of SCE, manufactured in-house, is used as a reference [−1 of PPy with 0.100 mol L−1 Tetrabuthylammonium hexafluorophosphate, TBAPF6, in anhydrous CH3CN. Electrochemical characterization of the p-doping/undoping process was carried out through 1000 successive voltammetric cycles (n = 1000) at a potential scan rate, ν, of 0.1 V s−1. The characterization as capacitor devices is made through analyzes of specific capacitance, retention capacity, and stability of the charge/discharge process in an aqueous system consisting of 0.1 mol L−1 of LiCl for 1000 successive cycles. All measurements were carried out under a high-purity argon atmosphere and at room temperature, 20 °C.The modification of ITO to prepare the ITO|PPy and ITO|PPy, PPy-nw electrodes is performed using past reported methods . Electroeference . PolymerC) for the bulk and nanostructured polypyrrole-modified electrode is calculated from cyclic voltammetry using Equation (1) [2E and 1E are the inversion potentials of the cyclic voltammetry, The value of V is the scan rate, and m is the mass of the polymer, which was calculated from the methodology described by del Valle et al. [C is the specific capacitance (F g−1), V is the potential range of the measurement, and m is the mass of the polymer. All electrochemical measurements were made using a potentiostat/galvanostat CH Instruments 900B .The value of the specific capacitance (tion (1) :(1)C=∫E1e et al. ,31. On te et al. :(2)C=itm−1 in a MultiRam spectrometer  with Nd:YAG laser as an excitation source (λexc = 514.5 nm), controlled by Opus software . All experiments were conducted at room temperature, in the air on thin films freshly deposited.The Raman spectroscopy of CP was measured in a range of 100 to 2000 cmThe morphology of the modified electrode was observed by scanning electron microscopy, SEM, initially depositing 5 nm of gold using sputtering and applying 10 KeV through an Inspect F50, FEI microscope .p-doping/undoping profile presents a rectangular wave, indicating ideal capacitive behavior [p-doping/undoping process of PPy bulk and PPy-nw is compared. PPy bulk presents a p-doping/undoping current density negligible compared to that of PPy-nw. This behavior is consistent with the significant increase in effective area attributable to the nanostructuring of the deposit on the same support area, similar to outcomes from different polymers in past reports [Voltammetric profiles corresponding to the electrosynthesis of PPy in the absence and presence of a template are shown in behavior ,34,35. T reports ,39,40,41p-doping/undoping CV profile in anhydrous conditions, p-doping process, as has been described in previous reports for analogous systems [The  systems ,40,42.p-doping and p-undoping charges is practically 1, implying that the nanometric morphology does not affect stability and/or durability of the conducting polymer film [The effect of electrode structure on the description of doping process is presented on mer film .−1, res since according to literature, these bands have been assigned to polarons and bi-polarons formation [Moreover, the Raman characterization of the deposits  shows thormation . In otheormation .−1 LiCl, Then, to check and compare the applicability of ITO|PPy bulk and ITO|PPy-nw as capacitors, their electrochemical response is analyzed in aqueous 0.1 mol Lp-doping/undoping processes in an aqueous LiCl-containing medium at different scan rates displays good stability and the desired rectangle shape. Current density attained by the nanowired electrodes is much higher than that from the bulk deposit. Additionally, relevant research has reported that when including nanostructures such as graphene oxide into electrode architecture, the current values attained were remarkably higher than regular PPy deposit [The CV response for the  deposit ,34. The  deposit  report sGalvanostatic measurements in an aqueous LiCl-containing medium, p-doping process, as already deduced from the data in On the other hand, when comparing the galvanostatic charge and discharge measurements of the modified electrodes , it is oFrom the potentiodynamic voltammograms and the galvanostatic potential/time transients at −1 for the PPy-Bulk and PPy-nw modified electrodes are 68.6%, 52.4%, 41.9%, 34.3%, 27.6% and 73.8%, 58.2%, 49.6%, 42.2%, and 36.3%, respectively, for 20, 50, 75, 100, and 150 mVs−1, indicating the better diffusion of ions in the nanostructure due to the decrease in the dead volume [For CV measurements, at lower scan rates, the calculated specific capacitance increases, and the electrode modified with PPy-nw presents a value 100 times greater than the respective electrode with PPy bulk. This is explained by the significant increase in active area of the nanostructured electrode on the same geometric area of the ITO support electrode. Additionally, the value of the charge decreases as the speed of the scan increases, which is related to the diffusion and migration of ions. However, the percentages of the specific capacitance with respect to that at 10 mVsd volume . From thd volume  when usiMoreover, galvanostatic measurements during 1000 cycles using PPy bulk and nw electrodes were also considered . In bothIf the specific capacitance is calculated after each galvanostatic charge cycle and presented with the capacitance retention  versus the cycle number, On the other hand, the evolution of capacitance retention is similar in both cases, indicating that nanowire morphology does not affect the stability of these device, but rather, it would only generate a significant increase in capacity. In parallel, this behavior is also observed in the charge and discharge measurements performed by cyclic voltammetry for 1000 cycles using an identical solution .Then, to corroborate the nanostructured morphology, scanning electron micrographs were obtained and analyzed, Finally, to evaluate the potentiality of the application of PPy-nw in the construction of supercapacitor devices The performance of the capacitors reported in the present work has comparable indicators with others from different authors. For example, the specific capacitance is at least 1500 times higher than that found for the graphene oxide PPy composite on glassy carbon electrode as from Yang et al. . On the On the other hand, capacitor stability is comparable to reports from other authors with similar materials. This behavior is attributed to the fact that the electrodes reported in the literature are additionally modified with GO, which aims to improve stability .In general terms, the outcomes from this study present a simple way to produce nanostructured supercapacitors based on electrosynthesized PPy nanowires with overall better performance when compared to similar devices from different authors. The methods are repeatable and may be applied to different polymeric systems , to compare and eventually optimize these results.ITO electrodes are modified in situ with polypyrrole nanowires by means of electrochemical steps. Cyclic voltammetry was used characterize the difference between bulk and nanostructured PPy by comparison of voltammograms during synthesis and doping processes; PPy-nw displayed higher currents and lower peak potential values if compared to bulk deposits, which translated on higher specific capacitance as well. Galvanostatic measurements in aqueous LiCl served to characterize the potential of the deposit in the design of capacitors. Nanostructured PPy showed 60 times higher specific capacitance, superior stability through 1000 charge/discharge cycles, and reproducibility compared to its bulk form. After analyzing SEM micrographs of the deposit, it was observed that PPy-nw deposit contain 15–30 nm width wire in a brush-like disposition normal to ITO electrode surface.The performance of the reported device is comparable to those found in literature from different authors with analogous systems. However, reports from other authors typically use graphene-based modifications, greatly enhancing the economic cost of the device in case of scalation to industrial prototypes. Consequently, the present outcomes were submitted as a patent application.The authors applied for an international patent called “Electrosynthesis of polymeric nanowires directly on solid surfaces (electrodes)”, with application numbers PCT/CL2018/050116 and reference 273176-WO.


Obesity is defined as a multifactorial disease, marked by excessive accumulation of body fat, responsible for compromising the individual’s health over the years. The energy balance is essential for the proper functioning of the body, as the individual needs to earn and spend energy in a compensatory way. Mitochondrial Uncoupling Proteins (UCP) help in energy expenditure through heat release and genetic polymorphisms could be responsible for reducing energy consumption to release heat and consequently generate an excessive accumulation of fat in the body. Thus, this study aimed to investigate the potential association between six UCP3 polymorphisms, that have not yet been represented in ClinVar®, and pediatric obesity susceptibility.rs15763, rs1685354, rs1800849, rs11235972, rs647126, and rs3781907 were determined by real-time Polymerase Chain Reaction (qPCR).A case–control study was conducted with 225 children from Central Brazil. The groups were subdivided into obese (123) and eutrophic (102) individuals. The polymorphisms UCP3 increase the risk for triglycerides, total cholesterol, and HDL-C levels. The polymorphism rs3781907 is the only one that could not be a biomarker for obesity as the risk allele seem to be protective gains the increase in Z-BMI in our pediatric population. Haplotype analysis demonstrated two SNP blocks  and (rs11235972 and rs1800849) that showed linkage disequilibrium, with LOD 76.3% and D’ = 0.96 and LOD 57.4% and D’ = 0.97, respectively.Biochemical and anthropometric evaluation of obese group showed higher levels of triglycerides, insulin resistance, and LDL-C and low level of HDL-C. Insulin resistance, age, sex, HDL-C, fasting glucose, triglyceride levels, and parents’ BMI explained up to 50% of body mass deposition in the studied population. Additionally, obese mothers contribute 2 × more to the Z-BMI of their children than the fathers. The SNP rs647126 contributed to 20% to the risk of obesity in children and the SNP rs3781907 contribute to 10%. Mutant alleles of UCP3 polymorphism and obesity were not detected. On the other hand, the studied polymorphism contributes to Z-BMI, HOMA-IR, triglycerides, total cholesterol, and HDL-C levels. Haplotypes are concordant with the obese phenotype and contribute minimally to the risk of obesity.The causality between  Obesity is a global health issue, affecting people of different age groups, sexes, and economic status with two-thirds of the obese population living in developing countries , 2. IncrObesity is a complex multifactorial disease. Several factors, including lifestyle such as eating habits and social behaviors, metabolic individual heterogeneity, psychological disorders, neurological changes, and individual genetic makeup are involved in the origin and maintenance of this disease throughout life , 6. It hThe fundamental cause underlying an obese phenotype is an imbalance between the amount of energy ingested compared to the amount of fat consumed by the body , 6, 9. AUCP3 gene, located at 11q13.4, has seven exons intervened by six intronic sequences. It is mainly expressed in both skeletal muscle and brown adipose tissue and the protein is related to cellular fatty acid metabolism. Upregulated expression of UCP3 has already been related to physical activity, fasting, and high-fat diet. On the other hand, downregulation has been reported to improve fat oxidative capacity [The genes encoding the human UCP are studied in association with obesity, as several gene variants have been related to hypertension susceptibility, diabetes development, and cardiovascular diseases . The UCPcapacity , 13. Whecapacity –162 per allele [UCP3 polymorphisms, the frequently studied SNP are rs15763, rs1800849, rs647126, rs7930460, rs1685356, rs1685354, rs11235972, and rs378190719.The gene variants most frequently used to investigate potential association with obesity susceptibility are Single Nucleotide Polymorphisms (SNP). To date, the risk alleles of about 750 loci of well recognized SNP with minimal allele frequencies as small as 1.6% have been contributing per-allele effects as low as 0.04 kg/mr allele . Moreover allele . Among UUCP3’s SNP that have not yet been represented in ClinVar® – a public repository which aggregates information about genotype–phenotype correlation and its relationship to human health – were used to investigate the potential association between UCP3 genomic variation and pediatric obesity susceptibility. From the 6 SNP, 4 were categorized as mutations affecting gene transcription, namely rs1800849 (upstream variant), rs15763, rs647126, and rs1685354 (downstream variants) and 2 were in the class of intron variants, namely rs3781907 and rs11235972. The study group comprised of eutrophic and obese children from Central Brazil.In the current study, a total of six The present observational case–control study included 225 children and adolescents, aged 5 to 19 years, of both sexes from Central Brazil. The participants were distributed as eutrophic or obese, corresponding to 102 and 123 people in each group, respectively. The study was performed following the ethical guidelines of the Declaration of Helsinki and was approved by the Ethics Committee on Human Research from the Pontifical Catholic University of Goiás under the registered number CAAE: 16303313.4.0000.0037. Written informed consent was voluntarily signed by parents/legal guardians of all participants.Participants were recruited from the Endocrinology Service at the Children’s Hospital . Laboratory analyses were carried out at the genetic laboratory of Replicon Research Group/School of Medical and Life Sciences from the Pontifical Catholic University of Goiás and Mutagenesis Laboratory from Federal University of Goiás. Malnutrition, overweight, severe chronic diseases, genetic syndromes, and the use of medications that affected body weight and mass deposition were defined as exclusion criteria.Anthropometric measurements including weight, height, and body mass index (BMI) of all participants and their biological parents were obtained by a pediatric endocrinologist with the aid of a fixed Tonelli E150-A stadiometer and a WELMY mechanical scale certified by the National Institute of Metrology, Quality and Technology (Inmetro). Inmetro is a governmental accreditation body responsible to maintaining the quality of instruments and pieces of equipment used in Brazil. The physician also performed a complete physical examination of the children and their biological parents. For all children, Z-scores of the BMI (Z-BMI) were calculated and interpreted according to the recommendation of the World Health Organization (WHO) . ChildreBiochemical tests were performed in an accredited laboratory in Goiânia, Goiás, Brazil. Blood samples were drawn by peripheral venipuncture following a period of overnight fasting ranging from 8 to 12 h. The assays included fasting plasma glucose, lipid panel, and insulin levels. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose levels were run in an Abbott Architect c8000® Chemistry Analyzer. Insulin levels were determined by immunoassay using an automated Abbott Architect i2000®.The reference values for fasting blood glucose are the same for adults and children. Thus, normoglycemic lays within the range of 3.3 and 5.6 mmol/L, pre-diabetics between 5.6 and 6.9 mmol/L and diabetics ≥ 7 mmol/L. Normal fasting insulin level is defined by the ranges 2.5 to 25 mIU/mL and < 15 mIU/mL for adults and children, respectively , 20. To Homeostatic Model Assessment Insulin Resistance) score was obtained using the equation: fasting serum insulin (μU/mL) x fasting plasma glucose (mmol/L)/22.5 [To assess insulin resistance in children, the HOMA-IR (/L)/22.5 .rs15763, rs1685354, rs1800849, rs11235972, rs647126, and rs3781907) , following to the manufacture’s laboratory protocol. The concentration of genomic DNA was determined using a spectrophotometer . Genotyping of the six SNP (p ≤ 0.05), considering confidence interval (IC) of 95%. Shapiro–Wilk’s and Levene’s tests were applied to check for normality and homogeneity of variance between groups before proceeding with additional analyses. Student’s t-test was used to compare the means of continuous variables between the studied groups. Pearson’s chi-square test (X2) was used to assess the deviations from Hardy‐Weinberg equilibrium and potential association between categorical variables, including the distribution of genotypes as a function of the risk allele between the obese and eutrophic groups. On the other hand, the standard McNemar test was used to determine if there were differences on the proportions of patients having wildtype alleles compared to the mutated genotype .Analyses were carried out in the SPSS® version 25.0  and Excel® version 16.62  utilities. In the current study, a significance level was set at 5%  were Z-BMI, HOMA-IR, triglycerides, total cholesterol, and HDL-C. As the reference category, fasting glucose was used as their means were not statistically different between eutrophic and obese participants. Therefore, it would be possible to test the association for a dominant model, where the presence of a risk allele would contribute to modulate the outcome variables in the whole group of participants.Multiple Linear Regression (MLR) was applied to observe whether the qualitative dependent variables could predict the outcomes of continues variables for the entire group. For the MLR the stepwise method was applied, and the equation was generated using beta standardized coefficients. Multicollinearity statistics was considered relevant to negatively affect the MLR model if tolerance was ≤ 20%. To test the assumptions’ compliances for MLR, the normality of the residues was verified according to the P-P plot graphs and the homoscedasticity by scatterplot. Furthermore, multinomial logistic regression (NR) was used to model the contribution of Haplotype block analysis was completed in Haploview® (Broad Institute) to test potential linkage disequilibrium (LD) for the studied SNP, using Gabriel’s confidence interval method . The funA total of 225 children and adolescents were enrolled in the study, including 123 (54.7%) and 102 (45.3%) participants in the obese and eutrophic groups, respectively. Regarding sex distribution, among the obese children, 60 (48.8%) were female and 63 (51.2%) were male, while in the eutrophic group, 52 (51%) were female and 50 (49%) males. Table It was observed that the mean values for weight, height, Z -BMI, mother’s and father’s, BMI, insulin levels, HOMA-IR, triglycerides, HDL-C, and LDL-C between obese and eutrophic groups were statistically different (p < 0.05). In the group of obese children, the mean value for HOMA-IR was 2.2 × higher than the mean of the eutrophic group, indicating insulin resistance among the obese children. Moreover, for the group of pediatric obesity, the mean of all variables revealed increased values but HDL-C, which showed an expected decrease. No statistical difference was observed for fasting glucose among the two groups. Table With respect to the normal distribution of data in the current study, few outliers were constant among all variables tested. However, Q-Q plots showed that skewness were observable only for the more extreme values related to outliers while most of the values for each variable remained adhered to the line of greatest fit as it would be expected if they truly came from a normal distribution. Consequently, because the data didn’t show heavy-tailed traits and because of the relatively large sample size of each group, non-normality presented no problems in hindering invalid further testing .p < 0.0001; R2 = 0.486), indicating that the predictors included in the model account for about 50% in the variability of Z-BMI in a pediatric population. The model only included in the equation variables that reached statistical significance:MLR was used to test if demographic, anthropometric, and biochemical independent variables could significantly predict the dependent Z-BMI for the whole pediatric population, consisting of obese and eutrophic participants. At first, it was identified and fixed multicollinearity among the predictors, which resulted in the removal of insulin , height (13.4%), weight (14.7%), total cholesterol (15.6), and LDL-C (16.3%) from the model. Thus, the MLR results from the whole set of participants showed a statistically significant model  between obese and eutrophic groups. The chi-square test also showed the frequencies of the genotypes in the studied population complied with the Hardy–Weinberg equilibrium (p > 0.05). When evaluating the genotypic distribution as a function of the risk allele, no statistical differences were observed Table . On the UCP3 gene were good predictors for the increase in the outcome variables. The multinomial logistic model was statistically significant  for all six SNP included in the study. Table UCP3 were implicated in predicting the outcome variables in a pediatric population. Two SNP variants were able to predict positive changes in Z-BMI (rs647126 and rs3781907), two variants associated with positive changes to HOMA-IR  while, on the other hand, two had a negative impact on HOMA-IR (rs3781907 and rs1685354). Additionally, positive changes in total cholesterol could be predicted by two variant genotypes (rs1800849 and rs11235972) and one SNP associated with positive increase of HDL (rs3781907). Finally, two SNP variants associated with positive changes in triglycerides plasma concentrations.A nominal logistic regression (NLR) was performed to verify if the genotypes aggregated for the risk alleles in the UCP3 gene at 11q13.4 genotyped in association with pediatric obesity in a cohort of children and teens from Central Brazil. The first three SNPS  were linked together in a separate haplotype block with a pairwise D’ value of 0.96, while a second block was inferred for SNP rs11235972 and rs1800849 with a pairwise D’ value of 0.97. Table With respect to haplotype frequencies, Fig. Understanding pediatric non-syndromic obesity requires the comprehension of the biological and environmental factors related to its development. Only after appreciating all the factors and covariables of such important heterogeneous, multifactorial, and complex disease that risk management could be planned to mitigate the health consequences for the susceptible population. For pediatric non-syndromic obese pollutions, clinical management has largely focused on metabolic concepts to cause or prevent severe outcomes. Scientific evidence has suggested that about 80% of obese children will become obese adults .UCP3 variants on the regulation of body fat deposition in an admixed pediatric population from Central Brazil.Genome-wide association studies (GWAS) with adult BMI and adiposity have associated many loci and thousands of variants with this phenotype. However, the contribution and role of those variants towards pathogenicity of obesity in adult and pediatric populations remains unanswered. Moreover, much less is known about the genetic variants associated with childhood obesity in the Brazilian population, well recognized for having extensive genetic admixture , 29. TheIn the current study, the sex of participants was not used as inclusion/exclusion criteria. Thus, random acceptance of both sexes led to the observation that pediatric obesity affected equally boy and girls. However, females seemed to be more susceptible to have larger Z-IMC than males as predicted by the Eq. 1.Our findings showed a set of covariables that better explains the variability of the Z-BMI in the pediatric cohort. Insulin resistance, age, sex, HDL-C, fasting glucose, triglyceride levels, and parents’ BMI explained up to 50% of body mass deposition in the studied population. One relevant observation regarding the contribution of parent’s BMI to Children Z-IMC favors previous observations that the combination of genetic, environmental, and lifestyle factors, such as the obesogenic environment a child is exposed to, contributes to the obese phenotype and, consequently, undesired health outcomes , 31. AddThe group of obese children showed higher levels of triglycerides, insulin resistance, and LDL-C and low level of HDL-C. Insulin resistance is related to the decrease of insulin capacity to uptake glucose  and it mUCP3 gene are responsible for interfering in energy homeostasis and related to cellular fatty acid metabolism. In the current study, the studied UCP3 polymorphisms showed the variants were not causative of pediatric obesity. The genotypic distribution of all 6 SNPS agreed with the ALFA database for the Latin America available at dbSNP/NCBI. On the other hand, the proportions between the risk allele were statically different between eutrophic and obese participants, except for the SNP rs647126. Thus, although the risk alleles were not causative, they contributed to the phenotype. The SNP rs647126 and rs3781907 did contribute to the increase of Z-BMI in our pediatric populations. The first contributed to about 20% to the risk of obesity in children while the latter contribute around 10%.In general, genomic variants in the UCP3 variants and HOMA-IR was found for 4 UCP3 SNP variants, namely rs647126, rs15763, rs3781907, and rs1685354, suggesting another important contribution to the UCP3 in obesity susceptibly due to insulin resistance and, consequently, higher blood sugar levels, that, in turn, it is a well stablished risk for T2DM. The findings from our study leads to future experimental designs to further understand the role of UCP3 in the development of T2DM. Moreover, increases risk for triglycerides, total cholesterol, and HDL-C levels were also associated with UCP3 mutant alleles. Although the increments were discrete, future studies would be welcome to shed some light into the role of UCP3 and cholesterol metabolism in humans (Table More importantly, the association between rs1800849 polymorphism is related to high levels of HDL-C, reduced chance to develop T2DM and decrease of BMI in adults [rs1800849 and BMI among Caucasian southern Italian children [The association of these polymorphisms with overweight, obesity, and BMI has remained controversial. Different studies suggest the wild genotype of n adults –46. A stchildren . A studychildren . On the children , 48. A Cchildren .Less is known about the rs11235972, rs647126, and rs15763 polymorphisms. A study by van Abeelen et al. reported an association between the mutant allele of the rs11235972 polymorphism and increased levels of CT and LDL-C . The rs6In the current study, rs3781907 polymorphism was the only one that could not be a biomarker for obesity as the risk allele seem to be protective against the increase in Z-BMI in a pediatric population Table , despiteUCP3 SNP in association with pediatric obesity in a highly admixed Brazilian pediatric population. The X axis indicates the relative physical position of each SNP within the gene. The haplotype block map for the whole span of the UCP3 gene, showing pairwise LD in D′, could be found in Table 2. Haplotypes and haplotype frequencies were shown in Table UCP3 variants with an obesity in a cohort of children from Central Brazil.Finally, Fig. UCP3 genetic variants on cohorts of obese Brazilian children make the current study innovative in the field. Finally, another limitation of our study relates to the lack of assessment regarding the potential obesogenic environment shared by the participant children and their parents.The discrete population size may be considered a limitation for the current study, even though participants were recruited following precise selection criteria for both obese and eutrophic groups. At the same time, the lack of research to understand the biological role of UCP3 SNP and obesity. However, the studied polymorphisms contribute to Z-BMI, HOMA-IR, triglycerides, total cholesterol, and HDL-C levels. Here we reported the presence of the risk alleles increased the chances of children having obesity and related comorbidities. Due to controversial findings about the role of UCP3 polymorphisms and obesity, further studies with a larger sample size and participants of distinct ethnic groups are required to confirm the potential of UCP3 variants to obesity in humans.In conclusion, the present study enrolling a cohort of admixed Brazilian children could not detected causality between the six We also highlighted the importance of management and follow-up of obese children to prevent metabolic disorders and comorbidity complications, such hypertension, T2DM, and cardiovascular diseases. The influence of the parents’ BMI on the Z-BMI of obese children reinforces the need for a careful look of this health problem under the assumption that childhood obesity is related to both genetic and environment factors. Thus, non-syndromic obesity should be managed and surveilled as a familial disease. The mitigation of deleterious health effects requires a holistic approach to guide the family to reduce their chance of undesired obesity outcomes. Unfortunately, however, for most non-syndromic obesity cases, a prognosis or a specific medical treatment/conduct protocols based on the contribution of multiple low risk alleles is not yet available, largely, due to the lack of knowledge regarding the multiple gene interactions and pleiotropic effect subjacent to complex traits, such as human obesity.


The site is demonstrated to retain deposits of Lower Palaeolithic artefacts, with 251 flakes, scrapers and cores identified to date. Infrared-radiofluorescence (IR-RF) dating of feldspar reveals 112 artefacts to have come from levels dating to at least 570 ± 36 to 513 ± 30 thousand years ago (ka) and are most plausibly assigned to an MIS 14 deposition, with artefacts produced during MIS 15 (approx. 560–620 ka). Indeed, these IR-RF samples provide minimum ages for artefacts. Combined with evidence from exposures linked to the original quarrying activities, a similar MIS 15 age is suggested for the more than 330 handaxe artefacts discovered in the 1920s. The remaining excavated artefacts come from levels dated to between 347 ± 22 and 385 ± 21 ka (MIS 10 or 11), with this later age interpreted to reflect post-MIS 14 deposition by substrate gullying and solifluction. These data demonstrate Fordwich to be one of the earliest Palaeolithic sites in northwestern Europe, and to retain the only large Acheulean handaxe assemblage directly dated to pre-MIS 13. Thus, Fordwich is determined to be a crucial piece of the pre-Anglian Palaeolithic puzzle in northern Europe.Northern Europe experienced cycles of hominin habitation and absence during the Middle Pleistocene. Fluvial gravel terrace sites in the east of Britain and north of France provide a majority of the data contributing to this understanding, mostly through the presence or absence of stone-tool artefacts. To date, however, relatively few sites have been radiometrically dated, and many have not been excavated in modern times, leading to an over-reliance on selectively sampled and poorly dated lithic assemblages. This includes Fordwich , where over 330 bifaces were discovered through industrial quarrying in the 1920s. Here, we present the first excavation and dating of artefacts discovered  Indeed, it is among the best known of the island's Lower Palaeolithic sites following the recovery of over 330 bifaces during commercial quarrying in the 1920s . FordwicAlthough thought to be ‘one of Britain's oldest sites’ , p. 8, pHomo antecessor, these 152 impressions were found in sediment associated with a ‘small’ collection of 78 flakes and cores  dated to greater than 800 ka currently represent the oldest evidence of hominins occupying Britain , High Lodge (Suffolk) and Waverley Wood (Warwickshire) (). 3.3Sixteen samples were taken for infrared-radiofluorescence (IR-RF) dating . The samet al. 5] and Brrcussion ,5,18, sorcussion . Some di. 6A century after Lower Palaeolithic artefacts were first discovered at Fordwich, the site is revealed to retain artefact-bearing gravels dating to approximately 542 000 years ago. These are among the oldest radiometrically dated artefact-bearing sediments in Britain and represent the fifth oldest archaeological occurrence currently known on the island . Further, we provide evidence that the 330 handaxes recovered from Fordwich during the 1920s are older than approximately 437 ka, and are most plausibly contemporary with the newly excavated MIS 14 sediments. Assuming an absence of occupation during the cold MIS 14 glacial stage ,34, FordNo handaxes were discovered during these new excavations, but they have revealed technological components previously unknown at Fordwich. We provide the first evidence for the presence of scrapers, with Smith , Ashmorein situ at the top of the lower main gravel (i.e. it is securely dated to approx. 542 ka). While it is easily differentiated from the chipped fluvial gravels at Fordwich, we remain hesitant in assigning it as a ‘miniaturized’  lithic ed’ , as is generally suggested for the Fordwich bifaces –7. Equalet al. [et al. [et al. [Prior to the recent discoveries at Fordwich, scrapers were unknown at the site ,5. Theiret al.  exposure [et al.  exposure [et al.  upper gr [et al. ,4. Our e [et al. ,18.. 6.1Until now it has been difficult to place Fordwich among the wider Acheulean of Europe. The French site of Moulin Quignon displays evidence of Acheulean hominins in northwestern Europe during MIS 17 ,34, and et al. [It is notable that the crude and highly rolled handaxes from Warren Hill conform with the elongated, thick, and poorly worked handaxes that characterize Fordwich –7,10,18.The 700 000-year-old Acheulean site of la Noira in Central France pre-dates Fordwich by two MIS stages and is one of the most technologically diverse pre-MIS 13 sites in Europe, providing a sample of bifaces, flakes, cores and retouched pieces . The tec. 7In 1981