In the past few years, much effort has been invested into developing a new blue economy based on harvesting, cultivating and processing marine macroalgae in Norway. Macroalgae have high potential for a wide range of applications, e.g. as source of pharmaceuticals, production of biofuels or as food and feed. However, data on the chemical composition of macroalgae from Norwegian waters are scant. This study was designed to characterize the chemical composition of 21 algal species. Both macro‐ and micronutrients were analysed. Concentrations of heavy metals and the metalloid arsenic in the algae were also quantified.The results confirm that marine macroalgae contain nutrients which are relevant for both human and animal nutrition, the concentrations whereof are highly dependent on species. Although heavy metals and arsenic were detected in the algae studied, concentrations were mostly below maximum allowed levels set by food and feed legislation in the EU.Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.This study provides chemical data on a wide range of algal species covering the three taxonomic groups  and discusses both benefits of and potential limitations to their use for food and feed purposes. © 2017 The Authors.  Macroalgae are part of the traditional food culture of many Asian countries, where they have been cultivated on a large scale for centuries. In contrast to Asia, the exploitation of this resource in Europe has been very limited and mainly focused on the industrial production of thickeners .Marine macroalgae can contain high concentrations of iodine.In the present study, we characterized the chemical composition of 21 species of marine macroalgae collected along the Norwegian coast, representing the three groups of red, green and brown algae. We also determined concentrations of the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg) and the metalloid As. We discuss differences among the species studied, assessing benefits of and limitations to their potential use as food and feed ingredients.et al.Macroalgae were harvested in October 2014 along the Northern coast of Norway (between 67.24 and 67.32° N and 14.47 and 14.72° E) in the intertidal or upper subtidal zone. Each sample consisted of pooled material of at least five individuals per species. The processing of the samples is described in detail in Biancarosa A complete list of the species identified in the current study as well as the sample locations is given in Table Dry matter (DM) content was estimated gravimetrically by freeze‐drying the samples at −20 °C in vacuum (0.2–0.01 mbar) for 24 h and then leaving them in vacuum at 25 °C until constant weight was reached.et al.Fatty acid (FA) composition was quantified by gas chromatography coupled with flame ionisation detection using a method described by Torstensen et al.Multi‐element analysis was carried out by inductively coupled plasma mass spectrometry (ICPMS) after wet digestion in a microwave oven, similarly to Julshamn et al.Inorganic As (iAs) was analysed by anion exchange high‐pressure liquid chromatography coupled with ICPMS (HPLC/ICPMS), based on Sloth et al.Iodine was quantified according to Julshamn et al.et al.Vitamin E forms were analysed by HPLC according to Konings n‐7), hexadecatrienoic acid (16:3n‐3), vaccenic acid (18:1n‐7) and α‐linolenic acid (18:3n‐3) in green algal species were 0.03–0.98, 0.01–0.18, 0.20–0.46 and 0.11–0.97 mg g−1 DW of total FAs respectively; while in red and brown algal samples these FAs reached concentrations of 0.01–046, <LOQ (below limit of quantification)–0.3, 0.01–0.16 and <LOQ–1.51 mg g−1 DW respectively , comprising mainly oleic acid (18:1n‐9) (0.48–20 mg g−1 DW). Concentrations of PUFAs were 2.1–2.3, 2–19 and 0.03–5 mg g−1 DW in green, brown and red algae respectively.Concentrations of palmitoleic acid  and Laminaria digitata (10 000 mg kg−1 DW).A detailed overview of the mineral composition of the algae in this study is presented in Table −1 DW) compared with red and brown algae (0.07–3.1 and 0.03–2.6 mg kg−1 DW respectively). The concentrations of Hg in the species studied ranged from <LOQ to 0.04 mg kg−1 DW . In this study, Pb was found to be low in red and brown algae (up to 0.58 mg kg−1 DW in Porphyra dioica) compared with green algae .The heavy metals Cd, Hg and Pb were found in all samples analysed, with their concentrations varying widely between species Fig. . The lev−1 DW) compared with red (6.4–24 mg kg−1 DW) and green (6.4–10 mg kg−1 DW) algae. Levels of inorganic As (iAs) were generally low in the species studied (mostly below 0.5 mg kg−1 DW)  in the samples is shown in Fig. α‐, β‐, γ‐ and δ‐tocopherol  compared with red and green algae, which contained only low levels of α‐tocopherol (10–26 and 8.8–12 mg kg−1 DM respectively). Tocotrienol was not detected or <1 mg kg−1 DW in all samples except for the brown alga H. siliquosa   and α‐linolenic acid (18:3n‐3).The FA compositions of the algae studied varied not only between the three phyla but also between different species belonging to the same phylum. This is consistent with previous reports and allows the FA profiles to be used for chemotaxonomic analysis to differentiate taxonomic groups.Porphyra species, where this marine omega‐3 fatty acid comprised more than a third of total FAs. High relative concentrations of this long‐chain (LC) PUFA have previously been reported in red algal species.n‐3 PUFAs owing to their low total lipid contents. Currently, the global recommendation for intake of EPA + DHA is about 200–250 mg day−1,In red algae, high relative concentrations of EPA  were observed, especially in n‐6 and n‐3 FAs in biological tissues is known to cause inflammatory processes in the body.n‐6 and n‐3 FAs is considered an index for evaluating the nutritional value of a dietary lipid source with respect to human and animal development and health.n‐6 and n‐3 is around 15–20:1 in Western dietsn‐6/n‐3 ratio of the algae in this study was within the recommended range of <5:1, they have the potential to enhance the nutritional quality of food products, e.g. by regulating low‐density lipoprotein and cholesterol levels, and thus may help to prevent inflammatory, cardiovascular diseases and nervous system disorders. Likewise, macroalgae with low n‐6/n‐3 ratio and high n‐3 LC PUFA contents could improve the FA composition of farmed fish species.An imbalance between P. dioica, U. intestinalis and Fucus vesiculosus reached 19, 29 and 30 g kg−1 algal DW respectively. This indicates that eating a 10 g portion of these dry macroalgae provides approximately 24, 36 and 37% respectively of the recommended daily intake of calcium for adult males and females in Nordic countries.−1 DW, the level of iron in the green alga U. intestinalis is higher than in many well‐known terrestrial sources of this mineral such as leafy green vegetables, legumes, nuts and cereal grains, which all contain between 2 and 4 mg iron per 100 g.Gadus morhua) having among the highest iodine contents (86 μg kg−1 wet weight (WW) on average).−1 DW, although variability among different species and phylogenetic groups  was considerable. The uptake of iodine in algae has been shown to be dependent on several factors such as salinity and temperature of the surrounding water, depth, and age of the thalli. The iodine values found in this study are in accordance with previous data on macroalgae collected in Norway and worldwideChordaria flagelliformis (1100 mg kg − 1 DW) and the kelps S. latissima (4600 mg kg−1 DW) and L. digitata (10 000 mg kg−1 DW). Since excess iodine can cause adverse health effects such as dysfunctions of the thyroid gland, dietary uptake of these algae may have to be limited.The species in this study were found to contain macro‐ and microminerals which are relevant for both feed and food purposes. For example, calcium in The heavy metals Cd, Hg and Pb were found in all species studied. Accumulation of these undesirable elements, naturally present in marine environments, can easily occur in marine organisms, including macroalgae.U. intestinalis (up to 3 mg kg−1 DW). Interestingly, Duinker et al.Ulva lactuca (0.18–0.23 mg kg−1 DW) collected in the south of Norway during spring/summer. Variability of metal levels in algae can be high among different species, seasons and collection sites.Ulva sp., with lowest metal concentrations in spring/summer and highest in autumn/winter.−1 WW respectively. These levels were not exceeded by any of the Pb and Hg concentrations found in the algae in this study (up to 0.3 and 0.01 mg kg−1 WW for Pb and Hg respectively).The concentrations of Hg were relatively low in all species studied, in line with previous findings.Arsenic in biological matrices exists either in organic forms (e.g. arsenobetaine and arsenosugars) or as iAs.H. siliquosa in which the concentration of iAs (2.4 mg kg−1 DW) reached 10% of total As. Data on concentrations of iAs in macroalgal species from Norwegian waters are scarce−1 (120 g kg−1 moisture content) for ‘seaweed meal and feed materials derived from seaweed’. This maximum level is set for total As, but authorities can request documentation showing that concentrations of iAs in feed materials are below 2 mg kg−1 (120 g kg−1 moisture content). In the current study, all species of red and green algae contained As concentrations below the EU current allowed level of 40 mg kg−1. However, four species of brown algae had levels of total As exceeding the maximum level allowed for algal feed materials, thus limiting the use of these algal species as feed ingredients in the EU.Levels of iAs in the species studied comprised overall <7% of total As; that is, As present in these macroalgae was found to be mainly in organic forms. Previous studies on As speciation have shown that the most abundant form of this metalloid in macroalgae is organic.α‐, β‐, γ‐ and δ‐tocopherol, while red and green algae contained only low levels of α‐tocopherol. The abundance of tocopherols detected in the present work was in accordance with earlier reports where brown algae were shown to contain higher levels of tocopherols than green and red algae.α‐, β‐, γ‐ and δ‐tocopherol range from a low level in the order Laminariales  to a high level in the order Fucales . The higher abundance of tocopherols in these species corroborates previous studies where the Fucales order seems to be unique regarding tocopherol composition.Brown algae had high contents of Poprhyra and Ulva species. These macroalgae could serve as good sources of high‐quality lipids and minerals. However, animal trials using seaweeds are needed to assess the bioavailability of these nutrients. Some of the brown algae in this study  contain high levels of arsenic and iodine, which could hamper their utilization for food and feed purposes. More data on seasonal and geographical variability are needed in order to assess the suitability of marine macroalgae collected in Norwegian waters for their use in food and feed.Based on our combined results on beneficial compounds and undesirable substances in the algal samples, red and green algae are the most promising algal groups for utilization in food and feed, especially Supplementary Table 1. Heavy metals and arsenic composition of 21 macroalgal species. Data represent mean values of two analytical measurements conducted on pooled algal material of several individuals per species. Data are expressed as mg/kg of the algal dry weight (dw). Values in brackets refer to concentrations of inorganic arsenic expressed as mg/kg of the algal DW.Click here for additional data file.


Peripheral nerve compression is reported to induce cortical plasticity, which was well pictured by former researches. However, the longitudinal changes brought by surgical treatment are not clear. In this research, 18 subjects who suffered from bilateral carpal tunnel syndrome were evaluated using task-dependent fMRI and electromyography assessment before and after surgery. The third digit was tactually simulated by von Frey filaments. The results demonstrated that the pattern of activation was similar but a decreased extent of activation in the postcentral gyrus, inferior frontal lobe, superior frontal lobe, and parahippocampal gyrus after surgery was found. The correlation analysis showed a significant correlation between the decreased number of activated voxels and the improvement of EMG performance. This result implied a potential connection between fMRI measurement and clinical improvement. Carpal tunnel syndrome is mostly characterized by sensory deficit of fingers. Differing from the traditional views, peripheral neuropathy, even the mild entrapment such as CTS, could induce cortical plasticity , 2. MostNapadow et al. assessed the functional and structural neuroplasticity in the somatosensory cortex when the fingers were electrically or mechanically stimulated , 3. The In this study, we tracked 18 subjects who suffered bilateral CTS for an average of 6 months. We aimed to explore the longitudinal changes of cortical plasticity utilizing fMRI. In addition, the potential connection between plasticity and sensory recovery was also followed with interest.A total of 18 subjects  who suffered from bilateral CTS, aged 40–50 years old with a mean age of 45.2 ± 3.3 years, were enrolled in this study. The age and severity of disease assessed by the Boston Questionnaire were controlled for better homogeneity (detailed information is provided in http://www.natus.com/). The data was recorded from a sensory conduction examination. A routine EMG test usually consisted of motor and sensory tests. For sensory conduction examination, one circle-like surface electrode, used as recording electrode, was placed at the level of proximal interphalangeal joint. Another stimulus electrode  was fixed on the wrist level between the palmaris longus and flexor carpi radialis. The third surface electrode, which was also circle like and used as reference electrode, was placed around the distal interphalangeal joint of the finger. The motor measurements were not involved in this research. One irrelevant technician completed all the EMG tests and then transported the sensory conduction data to the analyzer.We assessed the sensory function with EMG examination . The participants underwent a protocol including one task-dependent block-design BOLD sequence and a FSPGR 3D-T1 sequence. The first fMRI scan was performed before the surgery, and the second one was performed 6 months after surgery, which allowed enough time for cortical plasticity and avoided the interference of surgical incision-related pain. Considering subjects' individual arrangement, a 2-week variation was allowed.3), and number of acquisitions = 120.Foam padding was utilized to reduce head motion. For structural 3D-T1 imaging, we used the FSPGR sequence as follows: matrix size = 256 × 256, FOV = 256 × 256 mm, TR/TE = 8100/3.1 ms, FA = 8°, slice thickness = 1 mm, gap = 0 (isotropic voxel size = 1 × 1 × 1 mm), and TI (prepare time) = 450 milliseconds. For BOLD sequences, the parameters were as follows: sequence = GRE-EPI, interleaved scanning order, slice number = 43, matrix size = 64 × 64, FOV = 220∗220 mm, TR = 3000 ms, FA = 90°, slice thickness = 3.2 mm, gap = 0 (voxel size 3.4 × 3.4 × 3.2 mm2). Each session of the block design only involved a stimulus task of one hand. We utilized the models based on boxcar as our stimulus model during the task-dependent fMRI scan. We designed 6 cycles of ON-OFF. The whole scan starts with 12 seconds of dummy scan, which would automatically be discarded by the GE system. Then, an epoch of ON begins and lasts for 30 seconds. During this “ON” period, a 300 g von Frey filament was used to stimulate the finger pulp of the third digit of the subjects in a pseudorandom pattern, which in order to avoid sensory adaptation was brought by regular stimulus. Then, an epoch of “OFF” begins and lasts for 30 seconds, during which the stimulation was removed. One cycle lasts for 60 seconds and sequentially involves one ON and one OFF epoch. One complete session contains 6 cycles in all. The result of the first 3 cycles would be compared with the last 3 cycles in order to ensure uniformity. One single session of sensory stimulus task lasted for 6 min 12 sec in all. The whole scan of one subject mainly consisted of one 5 min-long FSPGR-T1 image and two 6 min 12 sec EPI block design images.Each participant underwent the same protocol. They were required to complete one sensory task, in which their digit 3 of both hands was stimulated using the 300 g von Frey filament  and across subjects. The homogeneity of the sensory task was controlled through utilizing a same technician and following a same protocol.During the OFF period, the participants were instructed to keep calm and avoid unnecessary movements in the MRI scanner. During the sensory stimulation, the participants were required to relax their bodies with no muscle contraction. The whole scan lasted for about 12 minutes.http://rfmri.org/DPABI) and SPM8 (http://www.fil.ion.ucl.ac.uk/spm/software/spm8/) was used for bold data processing. The first four dummy scans were already removed for signal equilibrium by the GE workstation. A slice timing procedure was applied for correcting the different acquisition times of each slice. Then, we realigned the functional volumes and corrected head motion by means of a six-parameter rigid body transformation [A toolbox including Dpabi  (http://p < 0.05 with FWE (family-wise error) correction. The significant activation area within groups through one sample t-test was reported and then binarized as a mask. A paired t-test was adopted to identify the difference of sensory stimulus response before and after surgery. The statistical comparison was carried out in the previous generated mask. The main analysis was performed within the boundary of the bilateral sensorimotor area. And the ROIs were extracted by WFU pickatlas tool (http://www.nitrc.org/projects/wfu_pickatlas/) and kept the same resolution and voxel size as the functional images.The statistical analysis of fMRI data uses a mass-univariate approach based on general linear models (GLMs). According to the experimental paradigm, we specified the GLM design matrix and estimated the GLM parameters using classical or Bayesian approaches. Then, we interrogated the results using contrast vectors to produce a statistical parametric maps (T maps in this study). The threshold was set at R square would be reported to indicate the goodness of fit.In addition to the fMRI analysis, we applied an extra correlation analysis of two measurements. The difference of both sensory latency and significant voxel number in the primary sensory area before and after surgery was calculated. And the ROIs were extracted by WFU pickatlas tool and kept the same resolution and voxel size as the functional images. A linear regression was done between the difference of voxel number in one hemisphere and the difference of sensory latency of the corresponding hand. The T-test showed significant improvement of sensory conduction test after surgery.The average median nerve sensory velocity of the right hand was 38.75 ± 7.71 m/s before surgery and improved to 52.35 ± 8.23 m/s after surgery, while the left was 35.03 ± 7.13 m/s and 50.43 ± 8.97 m/s, respectively, before and after surgery. The average median nerve sensory latency of the right hand was 7.87 ± 1.87 m/s before surgery and decreased to 4.55 ± 1.94 m/s after surgery, while the left was 8.35 ± 1.65 m/s and 4.93 ± 2.05 m/s, respectively, before and after surgery. The sensory threshold of the right hand was 0.37 ± 0.23 g and improved to 0.04 ± 0.02 g after surgery, while the left was 0.37 ± 0.24 g and 0.06 ± 0.08 g, respectively, before and after surgery . Paired t-test within group demonstrated that both presurgery and postsurgery groups showed significant activation in the postcentral gyrus. The pattern of activation was similar, but the extent of activation in the postsurgery group was smaller than that in the presurgery group  and dorsolateral prefrontal cortex. The PHC contributes to the network of brain regions participating in higher level cognitive processes, which involves visuospatial processing and episodic memory –28. The Another problem worth discussing is the selection of measurement in the correlation analysis. Napadow et al. discovered that the digit 3 projection area in the primary somatosensory cortex demonstrated more extensive activation when sensory stimuli were applied. They reasoned that the increased extent of activation in the primary somatosensory may be due to CTS patients' persistent paresthesia and pain, which represented increased afferent input for the affected digits . AlthougThe current study demonstrated a longitudinal change of sensory plasticity brought by surgical intervention. The changes of neuroplasticity were correlated with the improvement of symptoms. That revealed a potential connection between clinical outcome and cerebral functional reorganization.CTS patients showed a decreased extent of activation in the postcentral gyrus, inferior frontal lobe, superior frontal lobe, and parahippocampal gyrus after surgical intervention. The changes of activated voxels in the primary sensory area showed a significant correlation with the improvement of EMG performance. This study provided a longitudinal view of cortical plasticity brought by surgical decompression. And the result also implied a potential connection between fMRI measurement and clinical improvement.Although correlation analysis between voxel size and brain activity revealed significant results, this result was based on the data smoothed by a relatively large kernel. Magnitude of activity/deactivity affects the size of the area. Tests with much smaller kernel should be brought into consideration in smooth procedure.


UV) radiation‐induced reactive oxidative species is mainly responsible for the development of photoageing. Rosmarinic acid was one of the main bioactive components detected in Thymus vulgaris (TV) we extracted. In this study, UVB‐induced skin damages have been shown to be ameliorated by treatment with TV in hairless mice (HR‐1) skin, demonstrated by decreased matrix metalloproteinases (MMPs) and increased collagen production. However, the underlying molecular mechanism on which TV acted was unclear. We examined the photoprotective effects of TV against UVB and elucidated the molecular mechanism in normal human dermal fibroblasts. Thymus vulgaris remarkably prevented the UVB‐induced reactive oxygen species and lactate dehydrogenase. Dose‐dependent increase in glutathione, NAD(P)H: quinone oxidoreductase1 and heme oxygenase‐1, by TV was confirmed by increased nuclear accumulation of Nrf2. Furthermore, 5‐Methoxyindole‐2‐carboxylic acid was introduced as a specific inhibitor of dihydrolipoyl dehydrogenase (DLD). We demonstrated that Nrf2 expression was regulated by DLD, which was a tricarboxylic acid cycle‐associated protein that decreased after UVB exposure. Besides, TV significantly diminished UVB induced phosphorylation of mitogen activated protein kinases pathway, containing extracellular signal‐regulated kinase, Jun N‐terminal kinase and p38, which consequently reduced phosphorylated c‐fos and c‐jun. Our results suggest that TV is a potential botanical agent for use against UV radiation‐induced oxidative stress mediated skin damages.Solar ultraviolet ( Solar ultraviolet (UV) radiation consist of UVA 320–400 nm), UVB (280–320 nm) and UVC 100–280 nm), which induces cutaneous lesions such as photoageing and photo‐carcinogenesis. Particularly UVB is mainly responsible for the development of oxidative stress, inflammation and DNA damage resulting in photoageing, characterized by wrinkling, dryness, sagging and thickening 00–280 nm0–400 nm,Researches have been reported that accumulation of ROS could regulated the activation of antioxidant genes and transcription factors to counteract oxidative stress via varieties of cascades, such as mitogen activated protein kinases (MAPKs) and activated protein (AP‐1) signalling pathway Furthermore, UV‐induced oxidative stress is proved to regulate the phosphorylation of protein kinases Thymus vulgaris (TV) also called thyme is a traditional used herb rich in rosmarinic acid. The anti‐inflammatory, analgesic and antipyretic activities of Thymus serphyllum L. were evaluated in mice in vitrovia DLD‐Nrf2‐ARE, MAPK and AP‐1 signalling in normal human dermal fibroblasts (NHDFs).ELISA kits for MMP‐1, IL‐6, and TGF‐β1 were purchased from R&D Systems , while ELISA kits for procollagen type I were purchased from Takara . DMEM, FBS and penicillin streptomycin were purchased from Gibco BRL . MICA was used a specific inhibitor of DLD. Dry TV leaves were purchased from mountain rose herbs company . Unless otherwise mentioned, solvents were purchased from Samchun Chemicals , and inorganic salts were from Sigma‐Aldrich .The dried TV (10 g) were powdered and extracted three times with 1000 ml of ethyl alcohol (50%) for 24 hrs at room temperature. The ethyl alcohol was filtered and subjected to vacuum evaporation at 38°C.18 column . Elution was performed with a methanol/acetonitrile (3:1) gradient containing 1% formic acid. The gradient was linearly increased from 10% to 90% methanol over 35 min. The injection volume was 10 μl and the flow rate was 1 ml/min.High performance liquid chromatography (HPLC) was performed on a Dionex Chromelon TM chromatography data system with P580 and UVD100 detectors . Chromatographic separation was performed on a Waters Sunfire C2 UVB radiation seven times per week for the first week, and 100 mJ/cm2 three times a week for 4 weeks thereafter. However, the normal group was not exposed to UVB radiation.The animal experimental protocol for this study [KHUASP(SU)‐12‐09] was approved by the Institutional Animal Care and Use Committee of Kyung Hee University. Seven‐week‐old male albino hairless mice (HR‐1) (20–27 g) were obtained from Central Lab Animals, Inc. . UVB irradiation was performed as described previously Light‐bodied silicone  was introduced to obtain replicas of the dorsal skin. The visiometer technique was introduced to detect changes in the transparency of thin silicone replicas. Photos were obtained with a CCD video camera and analysed using Skin Viscometer SV 600 software . The R1, R2, R3, R4 and R5 values were obtained as previously described Biopsies  were obtained, fixed in 4% paraformaldehyde, dehydrated in ethanol and then embedded in paraffin. Approximately 10‐μm‐thick sections were deparaffinized and stained with haematoxylin and eosin and Masson's trichrome staining. Stained slides were then photographed using a light microscope .2. Then, sub‐confluent cells were subjected to irradiation supplied by a Bio‐Link BLX‐312  with 144 mJ/cm2 for NHDFs Normal human dermal fibroblasts  were cultured with DMEM (supplemented with 10% heat‐inactivated FBS and 1% penicillin‐streptomycin). The cells were maintained at 37°C in a humidified atmosphere containing 5% COCell viability tests were performed as described previously 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) was introduced to measure the antioxidant ability of TV extract. Ascorbic acid was used as a positive control.2) irradiation and samples treatment for 24 hrs, NHDFs were stained with 30 μM 2′7′‐dichlorofluorescein diacetate  for 30 min. at 37°C. The cells were then rinsed twice with PBS and subjected to a multi‐mode microplate reader (Molecular Devices Filter Max F5).Following UVB (144 mJ/cmThe lactate dehydrogenase (LDH) level in the culture medium was measured using an LDH cytotoxicity assay kit . This method is based on the LDH‐catalysed reduction in pyruvate to lactate by NADH. Briefly, equal amounts of culture supernatant were mixed with fresh LDH buffer containing NADH. After a 30‐min. incubation at room temperature, the absorbance was measured with an ELISA microplate reader at 490 nm.Total GSH content in the culture medium was determined using a GSH assay kit . The kit uses an enzymatic recycling method involving glutathione reductase to quantify GSH. The sulphydryl group of GSH reacts with DTNB  and produced a yellow‐coloured compound, 5‐thio‐2‐nitrobenzoic acid (TNB). The absorbance was measured with at 405 nm. The rate of TNB production indicates the concentration of GSH in the sample based on the standard curve provided with the assay kit.After exposure to UVB, NHDFs were cultured with MICA (1 mM) or TV (10 and 100 μg/ml) for 24 hrs. Total cell lysates were assayed by Western blot according to our previous research After exposure to UVB, NHDFs were treated with TV (10 and 100 μg/ml) for 3 hrs. The cytosolic and nuclear portions of the cells were separated with a commercial kit .We measured the concentrations of MMP‐1, MMP‐3, IL‐6, procollagen type I and TGF‐β1 in the medium with commercially available ELISA kits according to the manufacturer's instructions. Each sample was analysed in triplicate.At the indicated time, RNA from UVB‐irradiated NHDF cells was isolated using Trizol reagent according to the manufacturer's instructions . Reverse transcription (RT)‐PCR were performed in a Veriti Thermal Cycler  according to our previous research Total tissue and cell lysates were assayed by Western blot according to our previous research anova test. Statistical significance was set at P < 0.05.The data are presented as means ± S.D values of three independent experiments in triplicate. Statistical analysis was performed using one‐way To prepare TV samples, we extracted the dried seeds of TV 10 g) with 50% ethyl alcohol, obtaining crude product (1.46 g) with a 14.6% yield. As shown in Figure 0 g with Skin Wrinkle formation was measured in hairless mice chronically exposed to UV‐B radiation for 8 weeks. Integumentary images and cutaneous characteristics for wrinkle were taken and analysed by digital and CCD optical camera. Long‐term UVB irradiation accelerated wrinkle formation in the mouse skin, rendering the skin rough and scaly, indicative of skin photoageing. When TV (1% and 5%) was topically applied to the dorsal skin of hairless mice, the wrinkle formation was noticeably attenuated and the dorsal skin attributes were improved Fig. .Haematoxylin and eosin stained slides were photographed  and skin epidermal thickness was measured (Axio Vision Rel.4.8) Fig. . As showHistological staining with masson‐trichrome for dermal collagen fibres showed that chronic UVB irradiation damaged the cellular collagen density in the dermis. In contrast, there was a heavy staining of collagen fibres in TV (1% and 5%) treated hairless mice exposed to UVB, compared with that of UVB irradiated control group Fig. .As the structural protein of ECM, procollagen type I and elastin were degraded by MMP‐1 while stimulated by TGF‐β1. In our study, UVB irradiation caused 184.6% increase in MMP‐1 in the UVB irradiated group, compared with that of normal group. When TV 1% and 5%) was topically applied, the MMP‐1 level was alleviated in a dose‐dependent manner, which were 42.8.1% and 89.3%, respectively, compared with that of UVB irradiated control group , the impaired elastin was increased by 41.2% and 49.1%, respectively, compared with that of UVB irradiated group. In the case of TGF‐β1, the decreased protein was also significantly restored by topical application of TV (5%), which was increased by 39.4% compared with that of UVB irradiated control group  of TV extract, cell viability was reduced at higher concentrations (10 and 100 μg/ml), but there was not significantly difference compared with non‐treated group Fig. A.DPPH radical scavenging ability was measured to determine the antioxidant ability of TV. As shown in Figure It is well known that UV induces ROS, causing oxidative stress, which is harmful to cells. NHDFs exposure to UVB showed significant increase in ROS generation, whereas this trend was reduced by 40.8% when treated with TV (100 μg/ml) in UVB‐induced cells Fig. C. BesideTo investigate the role of DLD played in ameliorating UVB‐induced oxidative stress, MICA was introduced as a specific inhibitor of DLD. UVB irradiation impaired the DLD expression, while TV (100 μg/ml) enhanced its production by 24.6%, compared with the UVB irradiation control Fig. A. With tTo assess the antioxidant mechanism of TV acted, both cytosolic and nuclear Nrf2 proteins and antioxidants were assessed in UVB‐irradiated NHDFs. UVB irradiation induced the production of Nrf2 in nucleus. Simultaneously, nuclear amount of Nrf2 was significantly increased by 30.2% with the treatment of TV (100 μg/ml), compared with the UVB irradiation Fig. C. SimilaMatrix metalloproteinases is a major collagenolytic enzyme responsible for collagen damage in UV‐irradiated human skin On the other hand, this study attempted to confirm whether TV restored the collagen and TGF‐β1 breakdown of NHDFs caused by UVB. As expected, TV dose‐dependently enhanced the secretion of procollagen type I and TGF‐β1, which were dampened by UVB irradiation. Specifically, TV promoted the production of procollagen type I by 151.7% at 10 μg/ml and 158.4% at 100 μg/ml. In the case of TGF‐β1, those was 114.3% at 10 μg/ml and 128.6% at 100 μg/ml in NHDFs , the MMP‐1 transcript expression was sharply induced by 53.3% and 63.5%, respectively, compared with that of UVB irradiated control cells. On the contrary, the mRNA level of procollagen type I was diminished in the UVB induced NHDFs. With the treatment with TV (10 and 100 μg/ml), the expression of procollagen type I was meaningfully up‐regulated by 508.4% and 652.3%, respectively, compared with that of UVB irradiated cells Fig. .The MAPK signalling pathways was shown to mediate the expression of MMP‐1 Activated protein‐1 is the downstream factors of MAPK. To further investigate the action of TV worked, we measured the phosphorylated forms of c‐fos (p‐c‐fos) and c‐jun (p‐c‐jun) induced by UVB in NHDFs. As shown in Fig. , exposurBased on epidemiological studies, increasing numbers of people are suffering from skin damage because of overexposure to solar UV irradiation. Skin chronically exposes to solar UV radiation resulting in oxidative stress, inflammation, ROS‐mediated DNA damage, disorder of cellular signalling pathways, which accelerate skin photoageing Extracellular matrix is directly or indirectly disturbed by UV radiation with elevated MMPs activity. The MMPs activity was increased in skin because of UV irradiation, which resulted in collagen destruction and led to photoageing The level of ROS was dramatically elevated because of UVB induction, which was related to MMPs production and collagen fragmentation Ultraviolet induced ROS also accelerated the reaction of inflammatory cytokines. The cytokines, such as IL‐6, were proved to accentuate direct destructive effects on cells which accelerated photoageing via inhibition of p38‐MAPK signalling pathway in human keratinocytes via MAPK and AP‐1 signalling pathways. Taken together, inflammatory factor, MAPK and AP‐1 induction are initiated by oxidative stress Wrinkle occurs because of accumulated skin damages, such as matrix destruction and skin inflammation. Skin inflammation motivated MMPs, resulting in collagen degradation in the epidermis and dermis Ageing and many pathological processes have been known to be associated with the alteration of energy metabolism as a result of mitochondrial damages such as oxidative stress caused by ROS via inhibiting MMP‐1 expression. In addition, TV mitigated accumulation of ROS induced by UVB radiation. TV augmented production of DLD, which acted as cellular defense against oxidative stress through Nrf2‐ARE signalling pathway. Besides, TV inhibited the phosphorylation of MAPKs and AP‐1 signalling pathways in NHDFs. Based on our present results, we proposed the use of TV in curtailing skin oxidative stress caused by chronic UVB exposure.In conclusion, the present study demonstrated the photoprotective effects of TV on skin damage caused by UVB radiation. TV protected collagen breakdown The authors declare that there are no conflicts of interest.


Paspalum paspaloides, Murdannia triquetra, and Bidens frondosa dominated W and WB plots, and Microstegium vimineum and Bidens frondosa dominated D and DB plots. Abandonment of paddy fields led to a rapid decrease in soil organic carbon (SOC), and waterlogging accelerates SOC loss which should be attributed mainly to alteration of the vegetation cover. Six years’ rice cultivation increased SOC content by 13.5% (2.4 g kg−1) on average. In contrast, six years’ abandonment reduced SOC content by 14.5% (3.0 g kg−1) on average. Decline rate of SOC was 0.38, 0.64, 0.30, and 0.65 g kg−1 a−1 for D, W, DB, and WB, respectively. Such results indicate a significant risk of SOC loss from abandoned paddy fields.Paddy soils have been widely recognized as important carbon sinks. However, paddy field abandonment is increasing in the hilly area in subtropical China. Soil waterlogging and weed burning are common practices in abandoned paddy fields, which could affect vegetation cover and carbon sequestration. An rice cultivation experiment was ceased in 2006, and four new treatments were applied as waterlogging (W), drainage (D), waterlogging combined with burning (WB), and drainage combined with burning (DB). Waterlogging altered the vegetation cover and caused an associated change in biomass.  Wu analyzed soils sampled from some 2,700 sites under contrasting land uses from the 1980s to the 2000s, and concluded that rice paddy in subtropical China accumulated soil organic carbon (SOC) faster than other arable land and some woodland2. Rice cultivation can increase SOC even under unfertilized conditions. Using a long-term fertilizer experiment located in subtropical China, Shang et al. estimated that topsoil SOC sequestration rate in double rice paddy (two rice crops per year) was 0.96 t C ha−1 a−1 for the control and 1.01~1.43 t C ha−1 a−1 for the fertilizer plots3. It is estimated that SOC saturation value of paddy field in south China is 26~28 g kg−1 4. With an increasing rice productivity, paddy soils in subtropical China still have the potential to increase SOC.China is the largest rice producer in the world, with rice cultivation area of about 30 million ha in 2015, accounting for 18% of crop planting area across the country. Most paddy fields in China are located in subtropical areas with plenty of rainfall and radiation energy. Based on 404 observations across mainland China, Pan 5. Land use change directly alters vegetation cover and microenvironment, which further changes the quantity and quality of organic matter in soil that derives from plants as well as the decomposition rate of soil organic matter, resulting in changes in soil carbon pool9. In hilly regions, paddy fields are usually small and fragmentary, so the efficiency is low due to the difficulty to promote mechanical agricultural production. At the same time, China is undergoing a stage of rapid urbanization and many farmers gradually abandon such cropland and migrate to cities for higher-income jobs. Therefore, paddy field abandonment is increasing in hilly areas. Changes in SOC upon land use change may occur due to changes in carbon input and/or carbon decomposition rates10. In paddy ecosystems, substantial accumulation of SOC is attributed to high input of plant residues due to increasing rice production in recent decades and retarded decomposition under anaerobic conditions11. Increasing number of studies have reported that abandoned agricultural land will be occupied by natural vegetation and lead to SOC accumulation13. However, these studies focused on abandoned dryland. To our knowledge, change of SOC content in abandoned paddy fields in the subtropical area has been rarely reported. Shift from paddy fields to uncultivated land will result in dramatic change in soil environment. Field management, such as tillage, fertilization, irrigation, etc. will be ceased, and vegetation will be changed from crops to native vegetation. All these changes will lead to alteration in organic carbon input and decomposition, as well as other nutrient transformations15.Land use change is a major driving factor for the balance of SOC stocks and the global carbon cycle17. Additionally, the weed burning of aboveground weed residues may also influence the accumulation of soil organic carbon.Soil waterlogging and weed burning are common practices in abandoned paddy fields. In hilly regions, rice paddies are generally located in low-lying areas, and some abandoned paddy fields become waterlogged area due to poor drainage. Abandoned paddy fields are occupied by natural vegetation. And weed burning in winter is a common practice in abandoned paddy fields to avoid woody plant colonization, in case of reutilization of such land for crop land in the near future. Soil waterlogging and weed burning may alter vegetation cover in abandoned paddy fields and cause an associated change in belowground roots, which will affect organic carbon input. At the same time, soil water content will exert an impact on decomposition and mineralization of soil organic matter, thus further influencing the accumulation of soil organic carbonIn this study, we established an abandoned rice paddy experiment with four treatments, including waterlogging, drainage, waterlogging combined with burning, and drainage combined with burning. The objective was to examine the effects of soil waterlogging and weed burning on vegetation and SOC content in abandoned paddy fields.Paspalum paspaloides, Murdannia triquetra, Bidens frondosa, Microstegium vimineum, Nepeta fordii, Polygonum flaccidum, Cyperus iria, and Alternanthera philoxeroides are widespread species in the experimental plots. Waterlogging altered the vegetation composition. The dominant species were different between W and B as well as between WB and DB. Paspalum paspaloides, Murdannia triquetra, and Bidens frondosa dominated W and WB plots. Microstegium vimineum and Bidens frondosa dominated D and DB plots. Paspalum paspaloides and Murdannia triquetra are perennial hygrophyte; Microstegium vimineum is annual mesophyte; and Bidens frondosa is annual tropophyte. Vegetation compositions are similar between W and WB as well as between D and DB, indicating that weed burning had little effect on vegetation composition.Vegetation composition and Importance Value under different treatments are listed in Table −2 to 688 g m−2  on average in the twenty plots  on average in the twenty plots, which indicated that organic carbon input into the soil was far lower than SOC mineralization in the abandoned rice paddies. Weed burning had little effect on SOC content. In contrast with drainage, waterlogging accelerated SOC loss. SOC contents under W and WB were significantly lower than those under D and DB (P < 0.05). Decline rate of SOC was 0.38, 0.64, 0.30, and 0.65 g kg−1 a−1 for D, W, DB, and WB, respectively.In the six years before paddy field abandonment, rice cultivation increased SOC by 13.5%  and 2,000~3,000 kg ha−1 weed biomass  were incorporated into soil every year according to field observation. Also, a14C labeling study showed that a considerable amount of rice root exudation of photosynthesis-derived carbon (account for 8.0~19.3% of rice biomass carbon) was incorporated into SOC pool20. During the stage of abandonment, weed root biomass ranged from 2,500~6,000 kg ha−1  was way higher than that of the paddy field soil, even higher than SOC saturation content of the paddy field soil (26~28 g kg−1)24. The results suggest that whether SOC storage shows a positive or negative change depends on the capacity of an ecosystem to accumulate SOC when land use change occurs.Similarly, some researchers reported that land use change from paddy rice cultivation to grassland, woodland, and upland crop cultivation caused SOC lossIn the present study, waterlogging accelerated SOC loss, whereas weed burning had little effect on SOC loss. Possible reasons are ascribed as follows. First, waterlogging greatly changed vegetation composition Table . The domr = 0.697, P < 0.01, Fig. r = 0.590, P < 0.01, Fig. Correlation analysis showed that SOC decline rate was positively correlated with the initial SOC content (SOC content in 2006) . The region is characterized by a subtropical humid monsoon climate, with an annual average of air temperature, precipitation, sunshine, and frost-free period of 16.5 °C, 1,448 mm, 1,513 h, and 283 days, respectively. The region is featured with low hilly areas, with relative height less than 100 m. Gentle slope lands (6°~15°) account for 63% of total area. Many small and fragmented farmlands, accounting for 14%, are distributed on valley floor, the slope of which is generally smaller than 6°. The soil is developed from Quaternary red clay.A double rice (two rice crops per year) field experiment was established in randomized block design with five fertilization treatments and four replicates for each treatment in 1994. The soil has been subjected to paddy farming for more than 300 years. It was flooded rice cultivation with tillage for every cropping season. The fertilization treatments were NPK, NK, NP, PK, and control . Each plot was 7.5 m × 4.5 m in size. All the plots were arranged at a plane of the same altitude and surrounded by cement walls. The plots were not used for rice cultivation any more after the late-rice harvest in 2006. The twenty plots were divided into four groups, and each group comprised five plots which used to be one replicate of NPK, NK, NP, PK, and control, respectively. The four groups were arranged with four treatments which were waterlogging (W), drainage (D), waterlogging combined with weed burning (WB), and drainage combined with weed burning (DB). The five plots in each treatment was treated as replicates. Waterlogging meant collecting rainfall without drainage. The W and WB plots were usually saturated or flooded from April to August during which there was plenty of precipitation. In contrast, there was a water outlet in the cement wall for drainage, so the D and DB plots were non-flooded. Weed burning was implemented in December every year. The plant aboveground was burned to ash. Monthly averages of precipitation and air temperature from 2007 to 2012 were presented in Fig. 25. Three columns (20 cm length × 20 cm width × 20 cm depth) were collected in each plot to investigate the belowground biomass. Each column was split into 0~5 and 5~20 cm depths to measure the vertical distribution of weed root biomass. The samples were dried at 80 °C, and weighed to calculate their biomass.Vegetation investigation was carried out in July 2012 and July 2013. Three quadrats (1 m × 1 m) were set in each plot to investigate the height, density, coverage, and aboveground biomass for each plant. Dominant species were determined on the basis of Importance Value (IV), which was calculated as IV = (relative height + relative frequency + relative coverage)/326. Dichromate oxidation method has been widely used in soil investigations because of its simplicity and rapidity, although it is subject to interference by oxidizable or reducible soil constituents such as Cl−, Fe2+, and MnO226. Soil total nitrogen (TN) was determined by the Kjeldahl method27. Soil samples  contained 20.1 g kg−1 SOC, 1.78 g kg−1 TN, 0.76 g kg−1 total P, 15.2 g kg−1 total K, 14.9 mg kg−1 Olsen P, and 58.3 mg kg−1 available K in average. SOC decline rate (g kg−1 a−1) = (SOC2012 − SOC2006)/6. TN decline rate (g kg−1 a−1) = (TN2012 − TN2006)/6.Soil samples from the surface layer (0~20 cm) were collected in December in 2000, 2006, and 2012. Six soil cores were collected from each plot using augers and mixed to provide a single sample. The visible pieces of plant residues (>2 mm) were removed. Soil organic carbon (SOC) was determined by dichromate oxidation methodAll statistical analyses were performed with SPSS 17.0  using ANOVA, followed by the Tukey’s multiple range test, in which P < 0.05 was considered statistically significant. The correlation analyses were performed using Pearson correlation analysis.


People were categorized into tertiles of daily step count, and the 1st tertile had higher 24 h systolic BP, daytime systolic BP, daytime mean BP, and daytime systolic BP load than those in the other tertiles, even after adjusting for age and HbA1c. Participants with type 2 DM and low levels of physical activity exhibit higher 24 h and daytime systolic ambulatory BP values as compared with those who performed more steps per day, even after adjustments for confounding factors.The aim of this study is to evaluate the association between usual physical activity and 24 h blood pressure (BP) profile in people with type 2 diabetes mellitus (DM). This is a cross-sectional study of 151 participants with type 2 DM. Usual physical activity was assessed by step counting and self-reported questionnaire. BP was measured in office and by 24 h ambulatory BP monitoring . Mean participant age was 61.1 ± 8.4 years, 64% was women, and mean duration of diabetes was 14.3 ± 8.5 years. Ninety-two percent of participants had hypertension, and office BP was 138 ± 18/78 ± 10 mmHg. Inverse correlations were observed between step count and 24 h BP (systolic,  Hypertension is a major risk factor for the development and progression of chronic complications in diabetes mellitus. Sixty to 80% of persons with type 2 DM have hypertension , 2, and Current guidelines recommend that everyone who has no contraindications should engage in regular aerobic physical activity , but this suggestion is mainly based on benefits found in trials evaluating glucose control , 6. All However, a structured exercise program is not available to all patients with diabetes, and most of them will simply be advised to increase physical activity by their primary care physician. A home-based gradual physical activity program, designed to achieve a goal of 175 min of moderate intensity physical activity per week, was evaluated with other lifestyle changes as a cointervention  and founLow cardiorespiratory fitness, which is a hallmark of sedentary behavior, is a strong risk factor for mortality among males with diabetes . MoreoveAs 24 h BP profiles correlate better with outcomes than office BP, particularly in people with diabetes , 23, the2, physical disability preventing use of a pedometer, or participation in other research projects involving any type of intervention were excluded. The study protocol was previously approved by the Institutional Research Ethics Committee, and written informed consent was obtained from all participants.This was a cross-sectional study of 151 participants with type 2 DM recruited from the outpatient clinics of a tertiary center. The inclusion criterion was a diagnosis of type 2 DM . Persons with creatinine levels > 2.0 mg/dL, any disease that interferes with glucose control , unstable angina or acute myocardial infarction in the last 3 months, NYHA class III or IV heart failure, cirrhosis, alcoholism or illegal drug use, dementia, pregnancy or lactation, malignancies, BMI > 40 kg/mParticipants underwent an interview and clinical examination for collection of demographic and anthropometric data. Blood pressure was measured in office and by 24 h ABPM. The mean of two office BP measurements, determined in two separate days (one in the same day of ABPM placement) and obtained with a digital sphygmomanometer (Omron HEM-705CP Blood Pressure Monitor) on the left arm and with the person in the seated position, after a 5-minute rest, was considered for all analyses. Ambulatory BP monitoring was performed by the oscillometric method , with a 15-minute interval in the daytime and a 20-minute interval in nighttime periods. Participants were advised to maintain their usual daily activities. Sleep time was recorded as the period between the time when a participant went to bed and the time when the participant woke up in the morning. All ABPM measurements were obtained on a normal workday. The means of 24 h and daytime and nighttime systolic and diastolic BP were recorded, as well as BP loads (percentage of 24 h and daytime BP measurements ≥ 140/90 mmHg and nighttime BP measurements ≥ 120/80 mmHg). Hypertension was defined by the mean of office BP measurement ≥ 140/90 mmHg on two occasions or use of antihypertensive medications.Usual physical activity was measured objectively by step counting with a pedometer . Participants wore pedometers for 7 days, attached to the waistband of their clothing during waking hours, except when bathing or swimming. Participants were encouraged not to alter their usual physical habits during these 7 days. Every evening, participants recorded their number of daily steps in a diary and the pedometer was reset to zero to be worn on the next day. The pedometer also provides data on the daily distance walked in kilometers.Physical activity was also evaluated subjectively by self-reported levels of overall physical activity with the validated Portuguese-language version of the International Physical Activity Questionnaire (IPAQ long form) , and parFasting plasma glucose levels were measured by the hexokinase method, and HbA1c was measured by a high-performance liquid chromatography (HPLC) assay . Total cholesterol, HDL cholesterol, and triglycerides were measured by the colorimetric method, and LDL was calculated with the Friedewald formula. Creatinine was measured by the Jaffé method. Albuminuria was measured in a random sample, and a value greater than 17 mg/L was considered diabetic renal disease.Data are expressed as mean (±SD), median (interquartile range), or absolute and relative frequencies. Variable distributions were evaluated by a Kolmogorov-Smirnov test. Pearson correlation coefficients were computed between the number of steps/day and the BP variables. One-way ANOVA (Bonferroni as post hoc test) or Kruskal-Wallis test  was used according to the data distribution, and the chi-square test was used for comparison of categorical variables. Differences in office BP and ABPM values among categories of physical activity were evaluated. The sample was divided in tertiles of step count per day to better explore the association between this variable and BP measurements. The sample was divided also by the IPAQ results .P values < 0.05 (two tailed) were considered significant. Analyses were performed by SPSS version 18.0 or STATA 12.Sequential multiple linear regression models were constructed with office BP and ABPM values as dependent variables to adjust for possible confounding variables found on univariate analysis. A sample size of 150 individuals was required to detect a difference of 7 mmHg in systolic BP measured by ABPM , considen = 97 (64%); white, n = 116 (77%)) were included in the study. Mean age was 61.1 ± 8.4 years old, duration of diabetes was 14.3 ± 8.5 years, and BMI was 29.8 ± 4.7 kg/m2. Twenty participants (13%) were current smokers, 79 (52%) were living with a partner, and 67 (44%) had completed at least eight years of formal education. Mean HbA1c was 8.5 ± 2.0% (69 mmol/mol). Diabetes was managed with diet alone in 2 participants (1%), oral agents in 74 participants (49%), a combination of oral agents and insulin in 67 participants (45%), and insulin alone in 8 participants (5%). Of the 151 participants evaluated, 139 (92%) had hypertension; six were treated with nonpharmacological therapy alone, and 133 also with antihypertensive agents . Mean office BP was 138 ± 18/78 ± 10 mmHg.A total of 408 people were initially invited, and 257 people were excluded; 155 lived in other cities, 89 refused to participate, 12 dropped out, one died, and one presented a serious medical problem before the beginning of the protocol. Thus, 151 persons with type 2 DM  or office diastolic BP . However, significant correlations were found between step count and most ABPM parameters. Inverse correlations between daily mean step count and 24 h systolic BP , 24 h pulse pressure , and 24 h systolic BP load  were detected. There were no associations between step count and 24 h diastolic BP, 24 h mean BP, or 24 h diastolic BP load. Regarding daytime BP measurements, inverse correlations were found between mean step count and daytime systolic BP , daytime pulse pressure , and daytime systolic BP load . No correlations were observed between step count and daytime diastolic BP, daytime mean BP, and daytime diastolic BP load. Nighttime pulse pressure was the only nighttime BP variable correlated with mean daily step counts .The overall mean step count per day was 6391 ± 3357 , and participants walked a median distance of 3.1  km per day. No correlations were observed between step count and office systolic BP (p = 0.013) than those in the 3rd tertile, and HbA1c was higher in participants in the 2nd tertile than among those in the 3rd tertile (p = 0.008). No between-group differences were observed for the other clinical and laboratory parameters.Clinical and laboratory characteristics of participants according to the tertiles of mean step count/day  are listed in Participants with the least usual physical activity as evaluated by step count (1st tertile) had higher levels of 24 h systolic BP, 24 h pulse pressure, daytime systolic BP, and daytime pulse pressure than those with higher levels of usual physical activity (2nd and 3rd tertiles) . Mean daAccording to self-reported physical activity IPAQ, 136 (90%) participants were classified as active. However, only 18 participants (12%) reported regular planned physical activity in their physical activity log , comprising only 118 minutes of exercise per week on average . β = 6.40, 95% CI 0.31; 12.46, p = 0.040), 24 h systolic BP , daytime systolic BP , and daytime mean BP  values, but not with other variables, after adjustments , age, and HbA1c as independent variables. Less than 4873 of mean daily step count was associated with higher office systolic BP  and with perceived physical fitness  [In our sample, participants walked a mean of 6391 steps/day. This is consistent with previous studies, which have reported mean daily counts of 3448 to 7220 steps in persons with type 2 DM , 16, 25. = 0.02) . However = 0.02) –32.There are practical implications of the results presented herein. As previously described, traditionally used cutoff points of steps obtained from pedometers for classification of healthy adults are higher than those observed in this analysis. Thus, the traditional recommendation of walking at least 10,000 steps/day is probably a target difficult to attain for most individuals with type 2 DM , 26, 29.Usually, the cross-sectional design would be a limitation of this kind of study. However, as the information here obtained reflects real-life levels of usual physical exercise and BP profiles, we do not believe that it would be a major drawback. The main limitation of this report is related to an intrinsic characteristic of the device used to evaluate usual physical activity. Pedometers capture only movements of the lower body in the vertical plane and are unable to count other activities such as exercise involving upper limbs, as well as those performed on a bike or inside the water, like swimming.In conclusion, people with type 2 DM and low levels of usual physical activity exhibit higher ambulatory BP values in comparison with those who engage in more spontaneous exercise. Prospective cohort studies should be conducted to ascertain whether a causal relationship exists between usual physical activity and ABPM profile.


The use of agroindustrial by-products in ruminant nutrition to be an interesting alternative in order to reduce production costs and environmental impacts arising from the inadequate destination of residues. The initial step of beer production yields a large volume of wet brewer’s grains all year around, which is available at a low cost and has a high nutritional quality, and hence a big potential for animal production.Twenty-four Suffolk non-castrated male lambs, from simple parturition were kept in individual spots and allocated to four treatments constituted by four levels of substitution of sorghum silage by WBG . It was used roughage: concentrate rate of 50:50, based on dry matter.P ≤ 0.05). Substituion of sorghum silage by WBG did not change lambs’ feed DMI, daily weight gain and feed conversion.The ether extract intake increased while the acid detergent fiber intake decreased linearly (The substitution of sorghum silage by WBG as roughage showed to be a viable alternative from the productive and economic point of view for finishing of feedlot lambs. In the last 30 years, the main objectives of sheep production systems have changed from wool to meat production, due to consumer demands accompanied by animal breeding and nutritional improvements. The main meat productive category is lambs, which have high feed conversion, body weight gain and carcass quality, concomitantly with high nutritional requirements during the first months of life .Lamb feedlot fattening systems are an important alternative to stabilize meat quality and product offer throughout the year. Among other benefits to the production systems traditionally used, it may be highlighted the improvement on the animals sanitary condition, money turnover rate, uniformity of meat quality all year around, reduction of slaughter age and the availability of field and forage areas of pasture for the other categories of the herd .WBG) all year around, which is available at a low cost and has a high nutritional quality, and hence a big potential for animal production. This by-product has a high quality variation; the crude protein concentration varies from 170 to 350 g/kg dry matter –DM [NDF) from 550 to 650 g/kg DM [Agroindustrial residues are unconventional protein or energetic sources of great potential value for farmers in the effort to intensify and reduce costs, they also reduce the environmental impacts from the inadequate destination of residues . The beetter –DM  and NeutThis work was conducted with the objective of assessing the voluntary intake and meat production of intensive reared lambs fed with wet brewer’s grains as roughage feed in substitution of sorghum silage.UFSM) between October and December 2013. The experiment was approved by the Ethics Committee on Animal Use of the same institution (Protocol N° 037/2014). Laboratory analysis were performed at the Laboratory of Animal Nutrition .The work was performed at the Sheep Department of the Polytechnic School of Federal University of Santa Maria . After being weaned, animals were allocated to individual stalls (2 m2 each) within a shelter, with rice hull bedding, and equipped with individual feeders and drinking fountains. The lambs were allocated in a complete randomized experimental design (CRD) with four treatments, constituted by different levels of substitution of roughage (sorghum silage) by WBG  and six repetitions.Twenty-four Suffolk non-castrated male lambs, from single parturition were weaned with an average age of 60 days, properly dewormed  and inoculated against clostridium  at the The WBG used on the present study was acquired in a brewing agribusiness in Santa Maria and was preserved as silage. The diets were isonitrogenous and formulated according to NRC  in orderDM; Method 934.01 [OM; Method 942.05 [EE; Method 920.39 [CP = N × 6.25) was determined by Kjeldahl  and analyzed for dry matter  was assessed according to Method 973.18 of AOAC [ADL), bags containing residual ADF were treated with 12 M H2SO4 for 3 h , and ust et al. ). Acid d of AOAC ; both me of AOAC . For AciNFC; % in the DM) were computed as 100 – (CP + NDF + EE + ash) [TCH) as 100 - (%CP + %EE + % ASH). Metabolizable energy  was computed as ME = %TDN/(4.4 × 0.82), while %TDN = 31.4 + (53.1 x NEL) for silage [Non-fibrous carbohydrates (E + ash) , and Totr silage  and ME  for DM, OM, CP, EE, NDF, ADF, TC, NFC and TDN.P ≤ 0.05.Results were analyzed according to a completely randomized experimental design with four experimental diets, and six repetitions. Analysis of Variance and Regression Analysis were performed using SAS statistical package . DiffereThe WBG by-product had higher concentrations of CP and EE than Sorghum silage, but aNDFom, ADF and ADL concentrations were similar for both feeds Table . The che− 1 DM) was higher than that observed for 100% WBG inclusion ; Table P > 0.05) by WBG proportion in the final diet (Table c.a. 30% of DMI (2.7% BW) or c.a. 0.80% BW of NDF intake. Lack of differences were also reported in dairy cows by Geron et al. [In this study, voluntary feed intake was higher (4.12% body weight (n et al.  (inclusin et al.  and 10% n et al. . As DMI P < 0.001; Table Similarly, differences in chemical fractions intake were accounted for changes in feedstuffs composition. Hence that as WBG proportion increased EE intake was raised (Growth rate, feed conversion and body condition score did not change with rate of WBG inclusion Table  even whec.a. twofold that observed by McCarthy et al. [The positive response to WBG recorded in our experiment was y et al. , who wory et al. .The main objective of this work was to assess the voluntary intake and animal performance of intensive reared lambs fed with WBG in replacement of sorghum silage.The WBG had higher concentrations of CP and EE than Sorghum silage, and its chemical composition was typical of these brazilian by-products. Substituion of sorghum silage by WBG did not change lambs’ feed DMI, DWG and FC.


In the present investigation, we tested whether a personalized hydration strategy based on sweat rate would affect cardiovascular and thermoregulatory responses and exercise capacity in the heat. Eleven recreational male cyclists underwent two familiarization cycling sessions in the heat  where sweat rate was also determined. A fan was used to enhance sweat evaporation. Participants then performed three randomized time-to-exhaustion (TTE) trials in the heat with different hydration strategies: personalized volume (PVO), where water was consumed, based on individual sweat rate, every 10 min; ADL, where free access to water was allowed; and a control (CON) trial with no fluids. Blood osmolality and urine-specific gravity were measured before each trial. Heart rate (HR), rectal, and skin temperatures were monitored throughout trials. Time to exhaustion at 70% of maximal workload was used to define exercise capacity in the heat, which was similar in all trials (p = 0.801). Body mass decreased after ADL (p = 0.008) and CON (p < 0.001) and was maintained in PVO trials (p = 0.171). Participants consumed 0 ml in CON, 166 ± 167 ml in ADL, and 1,080 ± 166 ml in PVO trials. The increase in mean body temperature was similar among trials despite a lower increase in skin temperature during PVO trial in comparison with CON . HR was lower toward the end of TTE in PVO (162 ± 8 bpm) in comparison with ADL (168 ± 12 bpm) and CON (167 ± 10 bpm), p < 0.001. In conclusion, a personalized hydration strategy can reduce HR during a moderate to high intensity exercise session in the heat and halt the increase in skin temperature. Despite these advantages, cycling capacity in the heat remained unchanged.The optimal hydration plan [i.e., drink to thirst,  The aimEleven male recreational cyclists  were recruited. They had at least 3 years of experience with road or mountain bike cycling. Participants visited the laboratory on five separate occasions. They were asked to record their 24 h food intake and then to repeat the same intake before each visit. The study protocol was approved by the University Research Ethics Committee under the number 0015/250614. All participants gave written informed consent after being informed about the risks involved with the study. All procedures conformed to the code of Ethics of the Medical Association (Declaration of Helsinki).During the first visit to the laboratory, participants were asked about their normal hydration regimen during exercise to identify any habit that could help with the elaboration of the experimental hydration plan. Thereafter, semi-nude body mass  and height were recorded before we performed skinfold assessment  of the body composition to determine participants’ anthropometrical characteristics. Nine skinfolds  were assessed in triplicate by an experienced lab member and percentage body fat was estimated by using the equation proposed by Jackson and Pollock . Thereafmax) for 45 min to minimize learning effects throughout the study and to enhance their heat acclimatization profile. The two sessions were interspersed by at least 2 and at most 4 days to allow appropriate recovery. A fan was positioned at 120 cm of the front of the ergometer with a wind speed of 2.5 m/s to help increase sweat rate due to enhanced convective gradient  10 cm past the sphincter to monitor core temperature during each session. Skin temperature sensors  were placed on the chest, upper back, lower back, forearm, thigh, and calf. The sensors were maintained adhered to the skin, despite the presence of profuse sweating, by using a special tape permeable to air and moisture (Fixomull Stretch Tape). Skin temperature was calculated by using the equation proposed by Ramanathan . Mean bosumption . In the p < 0.05 from studies determining exercise performance . All data analyses were undertaken using SAS JMP PRO 13 and GraphPad Prism software.A sample size calculation was initially performed with G*Power software taking into consideration previously observed effects size and the requirements of a significance level of formance , 5. Normp = 0.801, F = 0.196] and despite the differences in fluid ingestion and body mass. Cyclists ingested 166 ± 167 ml of fluids in the ADL trial, which was much lower than the volume consumed in the PVO trial, 1,080 ± 166 ml, p < 0.001, t = 20.06, df = 11. Since we did not provide fluids in the control trial, the fluid consumption was 0. The volume of fluid ingested impacted the hydration status during exercise. In the CON and ADL trials, we observed a reduction in body weight , whereas in the PVO trial body mass did not change . The changes in body mass reflected the percentage of dehydration which was greater in CON and ADL in comparison with PVO  as described in Table p = 0.038, F = 4.70. Toward the end of the exercise trials, we observed a smaller increase in HR in the PVO trial (162 ± 8 bpm) in comparison with both CON (167 ± 10 bpm) and ADL (168 ± 12 bpm), p < 0.001, F = 229.5, as demonstrated in Figure During the incremental test, participants achieved a maximal power output of 372 ± 30 W. Therefore, participants workload during the trials was on average 260 ± 30 W. As described in Table The main goal of this study was to determine the impact of a personalized hydration strategy with water on cardiovascular, thermoregulatory and exercise capacity of recreational cyclists while exercising in the heat. Our main finding was that the personalized strategy resulted in lower HR and skin temperature during exercise in the heat and attenuated the degree of dehydration. Importantly, exercise capacity was similar regardless of the hydration strategy employed. These findings add relevant information to previous studies reporting conflicting results on the importance of hydration strategies on physiological outcomes involved in exercise performance in warm environments –4, 14.Hydration guidelines for athletes exercising in the heat encourage consumption of sufficient amounts of fluids during exercise to limit water and salt deficits and also to avoid over drinking , 16. Our2 = [stroke volume × HR] × O2 a-v difference) and frequently used to monitor cardiovascular response in exercise settings. Our results demonstrate an attenuated HR in the individualized, prescribed volume hydration strategy trial in comparison with ADL intake toward the end of the exercise trial. This occurred along with a lesser degree of dehydration observed in the PVO trial. The hypothesis that personalized strategy would improve endurance capacity was not confirmed in our study. Core temperature was similar regardless of the hydration strategy employed. Even though the total amount of water consumed was significantly higher in the PVO trial, both ADL and no fluid (control) trials elicited significant reductions in body mass that were still within the 2% threshold often considered to negatively influence performance  . While aIn conclusion, our study demonstrates that PVO hydration strategy was effective in reducing the cardiovascular stress and the increase in skin temperature imposed by a cycling session to exhaustion in the heat. Despite the lower cardiovascular and thermoregulatory stresses, the exercise capacity was similar regardless of the hydration strategy employed.This study was carried out in accordance with the recommendations of Federal University of Vale do São Francisco Research Ethics Committee. The protocol was approved by the #0015/250614. All subjects gave written informed consent in accordance with the Declaration of Helsinki.OL and JL conceived and designed the experiment. DM-M, AS-S, FF-J, and GS-S performed the experiment. DM-M, OL, and JL analyzed and interpreted the results. DM-M, AS-S, FF-J, GS-S, JL, and OL wrote and approved the final version of the manuscript.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.


The median FCd, TCd, UCd and BCd were 1.4 g, 1.4 g, 3.1 μg/g creatinine(cr) and 1.3 μg/L in control and 3.3 g, 3.6 g, 13.5 μg/g cr and 12.1 μg/L in polluted area. The 95% lower confidence bounds of BMD (BMDLs) of FCd for renal dysfunction were 1.36–1.55 g (BMR = 10%) and 0.88–1.11 g (BMR = 5%). The BMDLs of TCd were 1.29–1.46 g (BMR = 10%) and 0.73–0.95 g (BMR = 5%). FCd and TCd are valuable markers for the predication of renal dysfunction induced by cadmium. The BMDLs of FCd were close to previous report in Japan and the BMDLs of TCd were lower than the critical standard previously reported, in particular at BMR of 5% which can be interpreted as representing the influence of smoking.Recent several studies indicated that a more restrictive dietary intake guideline for cadmium should be made for sufficient health protection. In the present study, we showed the references level of food cadmium intake (FCd) and total cadmium intake (TCd) for renal dysfunction by using benchmark dose (BMD) approach. 342 subjects living in a control and a cadmium polluted area were included in this study. The FCd, TCd and cadmium in urine (UCd) and blood (BCd) were calculated or determined. Urinary β The kidneys are the main organs for Cd accumulation and are considered as the critical target organs of Cd toxicity. Previous studies have demonstrated that Cd in urine (UCd) and blood (BCd) are associated with renal dysfunction, in particular to renal tubular dysfunction4. The references level of UCd and BCd for renal dysfunction have been reported by using benchmark dose (BMD) approach7. However, only few studies in Japan have shown the references level of dietary or total Cd intake for renal dysfunction9. Cd contamination is reported in dozens of places in China11, and renal dysfunction has been observed in the residents living in those areas5. However, few studies have investigated the references level of Cd intake for renal dysfunction in China. In addition, the influence of smoking on the estimation of Cd references level has not been clarified.Cadmium (Cd) that widely distributed in the environment is one of toxic heavy metals. It can enter the human bodies through the food chain. Cadmium in the humans has a long biological half-life, 10–30 years14 to 10 μg/g creatinine for the occurrence of the classic tubular proteinuria15 have been reported. However, several studies have challenged the role of UCd as a potentially exposure biomarker18 because UCd is easily affected by physiological variations, such as diuresis. Dietary Cd intake might be a better marker than UCd12.The reference levels of UCd for renal dysfunction have been widely investigated. Thresholds of UCd, 2 μg/g creatinine for the onset of early renal damage20. In the current study, the reference levels of food Cd intake (FCd) and total Cd intake (TCd) for renal dysfunction were calculated by using BMD approach in Chinese population. Moreover, we also investigated the effect of smoking on the estimated reference level.BMD method has been used for estimation of reference levels of UCd, BCd and dietary Cd intake2Microglobulin (UBMG) between the polluted area and the control .Firstly, we showed the characteristics of study population Table . No signNext, we observed the association between FCd and UCd, BCd and UBMG Fig. . The UCdCorrelation analysis also showed that UBMG was positively correlated with the FCd, TCd, UCd and BCd (p < 0.05 or 0.01) Fig. . The corSubsequently, we evaluated the association between the prevalence of renal dysfunction and Cd intake Fig. . The datSubsequently, we calculated the BMD and their 95% lower confidence bounds (BMDLs) of FCd and TCd based on the elevated UBMG Table . The meaThe Cd concentration in tobacco in control area was significant lower than that in the polluted area (1.86 mg/kg vs 17.4 mg/kg) in our study. Next, we calculated the BMDLs of TCd hypothesizing that all the subjects smoke cigarettes containing similar concentration of Cd (1.86 mg/kg). Our data showed that no obvious decrease was observed in TCd compared with FCd  of 25 μg/kg body weight recommended by based on epidemiological studies . However, the BMDLs of TCd were 0.73–0.95 g. For 50–60 years old person, the tolerable month intake was about 1.12–1.4 mg/month which was lower than PTMI recommended by JECFA. Moreover, for those smoking population, much lower BMDLs were obtained compared with the total population. If we did not consider the contribution of smoking, the PTMI recommended by JECFA may sufficiently protect against Cd-induced renal effect. However, if the influence of smoking was considered, a stricter tolerable level should be recommended. In addition, the decrease of BMDLs in smoking population also indicates that those populations are susceptible to renal dysfunction.Previous study showed that the threshold values of lifetime Cd intake were 1.0 g in men and 0.8 g in women if considering the water intake24. For sufficient protection, a stricter dietary intake guideline should be established. A 25 μg/d (for 70 kg body weight) of PTWI was recommended by European Union25. In addition, if a more sensitive biomarker or earlier biomarker was used, such as urinary N-acetyl-β-D-glucosaminidase (UNAG), the calculated BMDLs may be much lower than the data above. Those data indicated that much effort should be made to control the maximally permissible concentration values for FCd as low as reasonably achievable26.Based on the BMDLs obtained in the present study, the exposure levels for some persons living in control area were also relatively high. UBMG was also positively correlated with TCd or FCd in subjects living in control area. Our data is in accordance with the findings that environmental cadmium exposure can cause renal dysfunction in the general population22. In our study, we also considered the contribution of smoke Cd intake to BMDL calculation. The BMDL of TCd decreased by 0.15 g compared with FCd which indicated that the tolerable levels should be much lower than the level recommended by JECFA if considered the contribution of smoking. However, the tobacco in polluted area contained a higher concentration of Cd than these in control or commercial cigarettes. Then we calculated the BMDL of total intake hypothesizing that the subjects in polluted area smoke the same cigarettes with control. The BMDL of total intake only slight decreased compared with food intake. Our data showed that the PTMI should be decreased by 1.4 μg/kg for a 60 kg of 50-year-old person. If the contribution of Cd intake from smoking to TCd was much lower than that from food, smoking may only slightly affect the BMDLs. However, if there are obviously differences in Cd intake from smoking, the contribution of smoke could not be neglected. We also estimated the BMDLs of TCd in the smoking subjects. The data indicated that a lower references level should be made for those smoking subjects than the total population.Cigarette smoking is another important exposure pathway of Cd in the general population. However, the contribution of Cd in tobacco was not considered in previous studies when calculate the BMDLs because the Cd uptake from smoking was small in subjects living in Cd polluted areasOur study has several limitations. First, the influence of age on BMDL calculation was not considered because the small population sizes. Second, it has been shown that there are differences in BMDL between men and women. However, no such calculation was performed in our study due to the small population size in control. Third, many biomarkers such as UNAG, Kidney injury molecule-1 can reflect renal tubular function. Only UBMG was adopted in our study. Finally, the absorbed cadmium may be more closely related with renal dysfunction than FCd or TCd. However, we did not estimate the cadmium uptake because the absorption rate of cadmium varies with the individual. Consequently, we did not calculate the BMD or BMDLs of cadmium uptake in this study.In conclusion, FCd is also a valuable marker for Cd-induced renal effects. The BMDLs of FCd for Cd-induced renal dysfunction was estimated to be 0.88–1.11 g (BMR = 5%) in Chinese population. The BMDLs of total Cd intake was estimated to be 0.73–0.95 g (BMR = 5%). The difference of 0.15 g was due to the influence of smoking.27. Briefly, two areas, located in Jiangxi Province, China, were included. A polluted area, named Dayu, was rich in Tin and tungsten. The mining of Tin and tungsten was begun from AD 660 and 1908, respectively. The rice fields were polluted with the wastewater. An average Cd concentration was 0.59 mg/kg in unpolished rice3. The drinking water was also polluted by wastewater. The Cd levels in vegetables were 0.06–0.66 mg/kg27. The previous studies showed that the daily Cd intake was about 299 μg/d in women and 313 μg/d in men28. We chose a non-contaminated area nearby as a control area. The Cd in polished rice was less than 0.08 mg/kg. The Cd levels in vegetables were ranged from 0.02 to 0.14 mg/kg. The total Cd intakes were 52.5–55.0 μg/d in men and 50.7 μg/d in women. The rice was the staple food. The people living in the two areas have common living conditions and lifestyles. More detailed information about the study areas and population has been reported in previous studies29.The detailed information has been reported in our previous study29.Total of 342 local residents (123 subjects in control and 219 subjects in polluted area), 173 women and 169 men, were finally included. Only the persons who have lived in the areas ≥25 years were included in this study. All participants completed two questionnaires. One was the weekly food consumption questionnaire, and the other was used for the collection of demographic information and smoking habits. All participants completed the informed consent. This study was proved by the Institutional Review Board of Fudan University, China, and all protocols were performed according to the approved guidelines. More detailed information could be found in the previous studies29. Briefly, the questionnaire of weekly food consumption was adopted to collect the type and amount of main foods consumed. 60 households were randomly collected from five villages in polluted area and two villages in control area. We mixed each types of food in same area together for chemical analysis. Nineteen samples of local tobacco were also collected. Then, after dried and washed, samples were dissolved in nitric acid for Cd determination by using flame atomic absorption spectrometry (AAS) or graphite furnace AAS . The following equations were used for Cd intake analysis: FCd = Σ (Cd in food × food consumption) + Cd in water × water consumption; TCd = FCd + Cd in tobacco × tobacco consumption × 10%. For estimation of Cd intake from food, the following age-related weighting factor was used during the calculation: 0–9 years, 0.41; 10–19 years, 0.89; 20–59 years, 1.000; ≥60 years, 0.82329.The previous studies have given the detailed information about the Cd intake estimation27. Briefly, the collectors were immersed in HNO3 solution (5%) for 24 h, and then they were washed with deionized water and dried. The blood and urine samples were stored at −20 °C in local laboratory. UCd and BCd were determined by using flame AAS after wet digestion27. We also performed quality control during the Cd determination30.The detailed information was described in our previous study28. The level of UBMG was determined by using radioimmunoassay (RIA) kit. Urinary creatinine (UCr) was determined by using Jaffe reaction method31. UBMG were adjusted with UCr and showed as milligram/g cr (mg/g cr). The cut-off values of 0.8 mg/g cr were used according to the 95th percentile of UBMG in the control (0.75 mg/g cr). UBMG levels >0.8 mg/g cr were regarded as “elevated.” Values below 0.8 mg/g cr were considered as “normal.”The detailed information was described in our previous studyhttps://www.epa.gov/bmds/what-benchmark-dose-software-bmds) to calculate the BMD and BMDLs. Benchmark response (BMR) of 5% and 10% were both used during the calculation. BMD and BMDLs were calculated using Gamma, Loglogistic and Logprobit models. We divided the subjects into four groups based on the FCd or TCd. Goodness of fit test was performed.We use the benchmark dose software  analysis. If P < 0.05 statistical significance was considered.SPSS 16.0  was used for statistical analysis. Data were listed as mean  or median . We used independent-samples t test or Mann-Whitney All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


To evaluate the capacity of OCT angiography (OCTA) for detecting infraclinical lesions in parafoveal capillaries in diabetic patients without diabetic retinopathy (DR).  This prospective observational cross-sectional case-control study analyzed the superficial and deep capillary plexuses (SCP and DCP) on macular OCTA scans (3 × 3 mm) centered on the fovea. We compared 22 diabetic patients (34 eyes included) without DR diagnosis on color fundus photographs, with 22 age- and gender-matched nondiabetic controls (40 eyes included). Qualitative analysis concerned morphological ischemic capillary alterations. Quantitative analysis measured foveal avascular zone (FAZ) size, parafoveal capillary density, and enlargement coefficient of FAZ between SCP and DCP. 2 in diabetic patients and 0.285 ± 0.150 mm2 in controls, P = 0.31. On the DCP, the mean FAZ area was 0.444 ± 0.153 mm2 in cases and 0.398 ± 0.138 mm2 in controls, P = 0.20.  Neither the qualitative nor quantitative parameters were significantly different between both groups. No microaneurysms or venous tortuosity was observed in any of the analyzed images. On the SCP, the mean FAZ area was 0.322 ± 0.125 mm OCTA did not detect infraclinical qualitative or quantitative differences in parafoveal capillaries of diabetic patients without DR in comparison with nondiabetic controls. Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in developed countries . By 2035OCTA is a recent noninvasive imaging technique that uses motion contrast to create retinal vascular networks images, by comparing the decorrelation signal among sequential OCT B-scans. In 2015, Spaide et al. demonstrated that OCTA could image separately each vascular layer of interest. The morphological retinal layer features in heathy eyes were described by Savastano et al. This type of specific analysis of either the superficial or the deep retinal capillary plexus or the choroidal vascular network is not possible with fundus fluorescence angiography , 8.OCTA has previously been used to describe retinal microvascular lesions in diabetic patients and mainly macular ischemic lesions –22. In dThis study aimed to evaluate if OCTA can detect infraclinical lesions in the parafoveal capillary network of diabetic patients without diabetic retinopathy (DR) on color fundus photographs. The main outcome measure was the presence of significant differences in qualitative and/or quantitative OCTA features in the parafoveal superficial capillary plexus and/or the deep capillary plexus between diabetic patients without DR and age- and gender-matched nondiabetic controls.In this descriptive prospective cross-sectional case-control study, 22 patients with type 1 or type 2 diabetes mellitus and with no clinical diabetic retinopathy (DR) nor diabetic maculopathy were examined. The study was conducted at the Department of Ophthalmology of the Creteil Hospital between March and September 2015. Twenty-two age- and gender-matched healthy control subjects, scheduled for regular visits, were also recruited during this time. Inclusion criteria were patients with type 1 or type 2 diabetes mellitus and the absence of diabetic retinopathy or maculopathy based on multimodal imaging performed on cases and controls by retina specialists . The absence of clinical signs of DR was confirmed by two combined examinations: fundus slit lamp examination and counter analysis of 30° color fundus photographs. A macular spectral domain OCT scan (SD-OCT) established the absence of macular edema. The exclusion criteria were as follows: refractive errors over 6 diopters in spherical equivalent, severe media opacities, macular edema, and other retinal diseases like age-related macular degeneration, hypertensive retinopathy, central serous chorioretinopathy, and vitreoretinal interface, choroid, or optic nerve diseases. Each patient underwent a questionnaire concerning basic demographics, ophthalmological history, and cardiovascular risk factors. We collected the following data: diabetes duration, glycated hemoglobin values, and glomerular filtration rate if available within the last month preceding inclusion. When the results were not available, new blood tests were performed. All patients underwent fundus biomicroscopy with slit lamp and digital color fundus photographs. As the current literature recommendations for DR screening , 27, 28,OCTA scans were acquired using a commercially available RTVue XR Avanti with the AngioVue OCTA software . The scanning area was captured in 3 × 3 mm sections, automatically centered on the fovea. The split-spectrum amplitude decorrelation angiography (SSADA) algorithm  revealedThree retina specialists examined OCTA scans for the qualitative analysis of parafoveal microvasculature. All patients' and controls' 3 × 3 mm OCTA scans were therefore arranged on a slide show to enable a magnified analysis. Thesuccession of images was randomly determined in order to avoid letting the readers know the diabetic status of patients. Each OCTA scan was evaluated next to the corresponding “en face” acquisition in order to distinguish vitreous condensations, motion or position artifacts from capillary loss. We retained the following abnormalities, commonly concordant with DR in literature , 30, 31:C. This C value was calculated by the difference between the FAZ area of both plexuses normalized with sFAZ: C = (dFAZ − sFAZ)/sFAZ. The vascular density of the parafoveal area was assessed by 2 different methods. With the first method, the “flow area” tool quantified the area of vessels, denoted A, in a 1.3 mm2 user-defined region of interest, centered on the fovea  with Angioanalytics quantification software of Optovue system. We measured three vascular parameters: FAZ area, parafoveal vascular density, and parafoveal intercapillary area. The “nonflow area” tool automatically defines borders of FAZ and measures its area Figures . The rea Figures .D, was then obtained with the formula D = A/(1.3 − FAZ). In this formula, D = vascular density of the parafoveal ring in the central 1.3 mm2, FAZ = area of the foveal avascular zone in mm2, and A = capillary flow area of the central 1.3 mm2, as shown in 2 was calculated by the difference between the parafoveal ring area and vascular area of the “flow” assessment tool with the formula PIA = 1.3 − FAZ − A, where FAZ = FAZ area (mm2) and A = vascular area of the central 1.3 mm2 (mm2).This selected area corresponded to the juxtafoveal region where capillary losses had been described in previous studies in patients without DR , 23. The2 test. The quantitative analysis compared each case to his age- and gender-matched control. Quantitative variables are described by their mean with standard deviation. Comparisons of means for paired variables were performed using the paired Student t-test. P values less than 0.05 were retained as significant. Analysis was performed with STATA version 13.0/SE . The coefficient regression values were calculated using the linear regression method. Multivariate analyses took into account all of the collected factors associated with the severity of the diabetic retinopathy [Qualitative variables are described in percentages. Comparisons of qualitative variables were performed using Mac Nemar's Chiinopathy : age, duWe examined 22 diabetic patients (15 women and 7 men) without diabetic retinopathy and maculopathy at fundus slit-lamp examination, fundus photographs, and Spectral Domain OCT. P = 0.05) and a higher body mass index (P < 0.001). There was no significant difference in other cardiovascular risk factors . Foveal retinal thickness was significantly thinner in diabetic patients . Choroidal subfoveal thickness was not significantly different between both groups: 264 ± 64 μm in cases versus 294 ± 68 μm in controls, P = 0.06, CI 95% , and is concordant with the mean choroidal subfoveal thickness described in the current literature for healthy adult subjects [Diabetic patients' clinical and laboratory findings are presented in patients : mean fosubjects .In the qualitative analysis, no venous tortuosity and no microaneurysm was found in neither plexus for both groups. No difference was noticed either when looking for irregular outlines and punched out borders of the foveal avascular zone (FAZ), as well as nonperfusion areas or morphological capillary network abnormalities .t-test comparing each measured parameter of diabetic eye to his matched control eye. As presented in 2 in diabetic patients versus 0.285 ± 0.150 mm2 in controls, with a mean difference of 0.037 ± 0.223 mm2, CI 95% , P = 0.31. The deep FAZ (dFAZ) area measured 0.444 ± 0.153 versus 0.398 ± 0.138 mm2 with a mean difference of 0.046 ± 0.221 mm2, CI 95% , P = 0.20. There was no association between the areas of sFAZ or dFAZ and the age, the glomerular filtration rate, the duration, and the control of diabetes on univariate and multivariate regression analyses. The coefficient of enlargement of FAZ was not significantly different between both groups . Parafoveal vascular densities were not significantly different between the two groups in both measuring methods. Their means were lower in the diabetic group except for the parafoveal density in the deep capillary plexus measured with the first method by comparison to controls  borders of the superficial capillary plexus (SCP) as well as uniform perivascular spaces . HoweverFAZ outlines can often be irregular in the absence of diabetes . Our quaP < 0.01, N = 7) [P = 0.04, N = 53 and P < 0.01, N = 24) [Though enlargement of FAZ in DR , 23, 24  N = 24) , 23. How N = 24) . Our resP = 0.14). Moreover, its mean value was higher in the control group with a mean of 0.60 ± 0.61 in cases and 0.43 ± 0.32 in controls. These results argue against the earlier alteration of deep vascular network in diabetic patients without DR. We noticed that this value was relevant only if sFAZ is not altered, as we observed it in our qualitative analysis.As previous authors suggested predominant and earlier enlargement of FAZ in the deep capillary plexus (DCP)  in DR, wThe development of DR can be seen either in a linear progression or in a stairstep model. This study could lead to hypothesize a stairstep model of the apparition of ischemic lesions on OCTA, concordant with the first microhemorrhages and microaneurysms on color fundus photographs. The literature also describes obvious vascular lesions with OCTA in mild diabetic retinopathy , 17, 18 The interpretation of our results should take into account that the area of the retina analyzed with 3 × 3 mm OCTA scans is limited and that the precise correlation of macular ischemia and peripheral DR manifestations remains to be established. Further studies using wider field OCTA, with peripheral fundus photographs are necessary to evaluate this correlation. In our study, a single examiner (MMG) performed FAZ measurements using an automatic program imbedded in the software of the OCTA. Literature argues in favor of the reproducibility and repeatability of FAZ measurement with the same method . The meaA noticeable characteristic of our patients is the short duration of diabetes (4.8 ± 4.0 years), with 6 patients examined at the discovery of the disease. Though it allows us to study the very beginning of the disease, we cannot exclude that capillary microchanges existing before clinical DR could not be described because of this relative short duration of diabetes.In conclusion, OCTA did not detect infraclinical quantitative or qualitative differences in parafoveal capillaries of diabetic patients without DR in comparison with controls. This result suggests that color fundus photographs and OCTA could concord to assess the absence of DR. The increasing distribution of OCTA in the clinical ophthalmological practice and the development of full-field OCTA fundus camera should be able to strengthen these findings on a wider scale. Considering that it is relatively easy to perform, inexpensive, and noninvasive, OCTA could become an efficient screening tool and might create a breakthrough in the solving of one of the major eye health problems in developed countries.