A number of studies have reported decreased white matter integrity in patients with schizophrenia, but little is known about the relationship between white matter integrity and antipsychotic medications.We enrolled 42 unmedicated patients (thirty were medication-naïve) with schizophrenia in a longitudinal trial with risperidone. Symptom severity was assessed with the Brief Psychiatric Rating Scale (BPRS). We obtained diffusion weighted images before medication was started, and after six weeks of treatment. Healthy controls matched 1:1 on age, gender, and parental socioeconomic status were also scanned twice six weeks apart. 30 diffusion sampling directions spanning the whole sphere were acquired twice and concatenated . After visual inspection of raw images we used TORTOISE for correction of bulk motion, eddy currents and susceptibility artifacts using a single interpolation step in DIFF_PREP. For each dataset, the first B0 image was selected as the reference for registration. Prior to registration, diffusion weighted and structural images were upsampled at a factor two and smoothed with a Perona-Malik anisotropic edge favoring gradient based filter to compute the transformations from moving to fixed images. After computation of transformations, original images were used to create the registered images. Bspline correction was done with the T2 weighted image (approximated from a T1 image using AFNI’s FATCAT). Diffusion and structural images were resampled to 1.5mm isotropic voxels. Gradient tables were rotated along with motion correction. To obtain a summary measure of motion, the root-mean-square (RMS) was calculated both for absolute (RMSabs) and relative (RMSrel) movement. Datasets with RMSabs of greater than the voxel edge length were excluded from further analysis. Tensors were computed with DIFF_CALC using a linear fitting algorithm. To spatially normalize diffusion images to the Illinois Institute of Technology atlas (IIT2) space, we implemented an optimized non-linear image registration using a modified version of 3dQwarp in AFNI. The warping optimization implements an iterative refinement, where an input image is repeatedly processed through an optimizer in smaller and smaller patches, incorporating convergence criteria at each patch level to better resolve artifacts, with a final patch size of 3x 5x 3 mm. To assess whole brain voxel-wise group differences and changes over time in scalar indices used AFNI’s 3dttest++  with clustsim, a bootstrapping method used to correct for multiple comparisons.Mean age of patients was 26.62 years, 62% of subjects were male. Of the 42 patients included here, 33 completed the study. BPRS total scores decreased significantly after six weeks of treatment, average risperidone dose at that time was 3.73+/-1.72mg. Fractional anisotropy (FA) was significantly decreased in a small area of the medial temporal lobe and mean diffusivity (MD) was significantly increased in the hippocampal part of the cingulum in unmedicated patients (n=40) compared to healthy controls (n=41). Longitudinal analyses showed no changes in FA, MD, RD or white matter macrostructure in healthy controls over time, and no changes in patients after six weeks of treatment with risperidone.With state of the art data-processing methods we only found small areas of white matter integrity deficits in our predominantly medication-naïve patients. This is consistent with prior reports of limited white matter alterations at disease onset that may progress with illness duration. Our data suggests that a short-term course of antipsychotic medication may not alter white matter microstructure, but studies with longer follow up durations will be important to determine long term effects of antipsychotic medications.


Lumbriculus variegatus and Asellus aquaticus). This effect appeared to be overruled in the field due to unknown sources of natural variation. Although we observed higher consumption rates in species-rich ditches compared to species-poor ditches, consumption rates were comparable for all three types of DECOTAB deployed. Upon reduced food quality and palatability, results presented here hint that PUFA like linoleic acid may be a key OM attribute driving the performance of benthic macroinvertebrates and inherent functioning of aquatic ecosystems.Chemical composition of organic matter (OM) is a key driver for detritus consumption by macroinvertebrates and polyunsaturated fatty acid (PUFA) content is considered a candidate indicator of food palatability. Since traditionally used complex natural OM covaries in many quality attributes, it remains uncertain whether benthic invertebrates developed an actual preference for PUFA-rich food. Therefore we aimed to test the influence of the PUFA linoleic acid on OM consumption by aquatic macroinvertebrates using standardized surrogate substrates  with added linoleic acid (PUFA) in comparison to consumption of DECOTAB containing only cellulose (Standard) or ground macrophytes (Plant). In microcosms, we observed a higher consumption rate of PUFA DECOTAB in comparison to Standard DECOTAB in two functionally distinct invertebrate species ( Dead organic matter (OM) fuels benthic food webs1357The chemical composition of OM is a key driver that can either promote or retard OM-degradation and the availability of newly produced, high quality OM can cause strong growth responses in invertebrates1112131413161821231314233031Lumbriculus variegatus and the shredder Asellus aquaticus) in laboratory microcosms and to natural invertebrate communities in lowland ditches with either a species-rich (>10 taxa) or species-poor (<5 taxa) community composition.Since it can be expected that consumption of OM by species-rich detritivorous invertebrate communities more strongly depends on OM quality compared to consumption by species-poor communities due to preferences of individual species for higher quality detritus sources, we hypothesize that addition of linoleic acid to standardized OM . The shredder A. aquaticus consumed all DECOTAB types significantly faster than the deposit feeder L. variegatus . Consumption rates were significantly different between species and DECOTAB qualities , Chironomus spp. (9.1) and Gammarus pulex (4.6). Comparable macroinvertebrate numbers were collected from cages in both type of ditches, however, a significant difference in functional feeding composition of the macrofaunal communities was observed between species-rich and species-poor ditches . Species-poor ditches were characterized by a dominance of deposit feeders (41.8% of the community versus 20.7% in species-rich ditches) and low representation of shredders . Scrapers occurred in comparable densities in both types of ditches . Predators and filter-feeders occurred in low densities.From the cages, almost 1700 macroinvertebrates were collected and identified, per cage on average 31 ± 3 individuals (mean ± SE) belonging to 4.9 ± 0.2 taxa. The dominant taxa were 1,47 = 16.1, P < 0.001). Degradation of Standard DECOTAB  was significantly slower than degradation of Plant DECOTAB , while for PUFA DECOTAB degradation  was intermediate in species-rich ditches . Microbial decomposition of DECOTAB (fine mesh cages) was comparable between ditches with different species richness (P = 0.78), but was significantly different between DECOTAB qualities  than decomposition of Plant DECOTAB (78.6 ± 6.6 mg d−1). Macroinvertebrate consumption was significantly higher for all DECOTAB qualities in the species-rich ditches compared to the species-poor ditches , but consumption was similar for all DECOTAB qualities within each ditch type  after 43 days was different between ditch species richness and between DECOTAB qualities. Degradation in species-rich ditches was faster compared to species-poor ditches  in comparison to cellulose only OM (Standard DECOTAB), indicating that the addition of linoleic acid to food sources improved the OM quality for the detritivorous macroinvertebrates tested in this study. The observed importance of PUFA content for consumption rate corroborates previous research in the field, where PUFA content was shown to be a good indicator of food quality for invertebrates142313A. aquaticus consuming all DECOTAB qualities significantly faster than the deposit feeder L. variegatus, we observed large differences in the relative influence of litter quality on consumption rates by both detritivorous species. This suggests that the significance of PUFA on OM-consumption is different between species. In comparison to consumption rate of surrogate litter consisting of only cellulose (Standard DECOTAB), surrogate litter consisting of cellulose and additional 5‰ dry mass linoleic acid was consumed by A. aquaticus at 163% and by L. variegatus even at 652%. For A. aquaticus, the consumption rate of linoleic enriched standardized OM (cellulose) was around 60% of complex plant OM (Plant DECOTAB), while for L. variegatus the addition of linoleic acid to standard OM resulted in comparable consumption rates (~90%) compared to consumption of complex plant OM. The latter suggests that linoleic acid is an important component governing OM consumption by L. variegatus.In our laboratory experiment we tested two functionally distinct invertebrate species for their consumption rates of different quality food sources. Besides expected differences in consumption rates between species, with the shredder Although we only tested two species that represent two different feeding habits, it can thus be speculated that different responses to food quality between functional feeding groups can have impact on organic matter processing by the detritivorous macroinvertebrate community. Detritivorous macroinvertebrates belonging to different functional feeding groups, e.g. shredders and deposit feeders10In this study we observed higher consumption rates in lowland ditches with a more diverse macroinvertebrate community, comparable to various aquatic systems in which species richness and trophic diversity positively affected decomposition7A. aquaticus between seasons, due to season-specific physiological statusOne of the sources of natural variation may have been the availability of other food sources which could have influenced consumption of the introduced artificial substrates at the ditch scale. Detritivorous macroinvertebrates experience large shifts in food quality over the seasons37In conclusion, by using surrogate litter in which we manipulated PUFA content and food palatability, we observed a strong influence of the presence of the PUFA linoleic acid in food items on OM consumption rates by aquatic invertebrates in our laboratory microcosms. Observed differences in consumption rates between the two species are most likely caused by differences in feeding mode and preferences for OM in different stages of decomposition. However, the observed influence of linoleic acid on OM consumption was overruled in a natural environment, most likely caused by heterogeneity in invertebrate community composition and other sources of natural variation. Results presented here thus hint that omega-6 PUFA like linoleic acid may be a key OM attribute driving the performance of macroinvertebrates in benthic environments where food palatability is drastically reduced.et al.Potamogeton pusillus and Myriophyllum spicatum to represent a natural litter source. In order to minimize microbial resources as potential food source in the Plant DECOTAB, fresh plant material was collected, rinsed and dried at 60 °C. Plant DECOTAB contained the most balanced food source including a wide range of PUFA16Surrogate litter sources that have traditionally been used to study invertebrate consumption are difficult to manipulate chemically to study effects of specific compounds29302∙2H2O, 90 mg MgSO4∙7H2O, 50 mg NaHCO3 and 10 mg KHCO3 dissolved in 1 L demineralized water . We determined the consumption rates by the deposit feeder L. variegatus (laboratory culture) and the shredder A. aquaticus (collected from a nearby ditch and adjusted to the climate chamber for seven days) for each of the three DECOTAB types. We used allometric relationships for the invertebrates to standardize invertebrate biomassA. aquaticus  or twelve L. variegatus  were added to each microcosm at the start of the experiment. For all combinations of macroinvertebrate species and DECOTAB types we set up five replicate microcosms containing three DECOTAB. As a control for leaching and abiotic degradation, five control microcosms per DECOTAB type were included containing no detritivores. Microcosms were placed in a climate chamber  and were continuously aerated. DECOTAB were collected after sixteen days, carefully washed, dried in a stove  and weighted.Microcosms were created by adding a layer of 1 cm pre-annealed  quartz sand to a glass jar (100 ml) and adding 95 ml of Dutch Standard Water , consisting of 100 mg CaClDrainage ditches are a common aquatic habitat in the lowland agricultural landscape of north-western Europe and can be a significant habitat type for aquatic invertebratesTo facilitate retrieval in the field, we deployed cages  containing five DECOTAB of a single type closed off by either fine mesh (width 51 μm) to quantify decomposition by micro-organisms (mass loss by leaching of compounds from the agar matrix under controlled conditions was negligible), or coarse mesh  to quantify consumption by macroinvertebrates. On 14 April 2014 at each of the six ditches (depth 0.25 to 0.65 m), three coarse mesh and one fine mesh cage per DECOTAB type were placed on the sediment alongside the bank in a randomized order at 2 m intervals. After 43 days of exposure, the cages were carefully collected from the ditches and each cage was placed in separate bag including the macroinvertebrates that had colonized the cage. DECOTAB were rinsed and subsequently dried in a stove  and weighted. Consumption by macroinvertebrates was calculated by subtracting mass loss of DECOTAB in fine mesh cages from mass loss of DECOTAB in the coarse mesh cages.Since macroinvertebrate distribution in these lowland ditches is very heterogeneousA. aquaticus and L. variegatus for each DECOTAB quality in the microcosms were analysed using ANOVA and post-hoc Tukey’s b test. From the field data, we analysed DECOTAB degradation (i.e. loss in coarse mesh cages), microbial decomposition (i.e. loss in fine mesh cages) and macroinvertebrate consumption (i.e. degradation minus decomposition) per DECOTAB quality and ditch species richness using two-way ANOVA and post-hoc Tukey’s b test. Data were analysed for homogeneity and transformed when necessary, data analysis was performed in IBM SPSS Statistics 20. Differences in the functional composition of invertebrate communities were tested with a Gower-based ANOSIM47Consumption rates of How to cite this article: Vonk, J. A. et al. The significance of linoleic acid in food sources for detritivorous benthic invertebrates. Sci. Rep.6, 35785; doi: 10.1038/srep35785 (2016).


Secondary lymphedema, a life-long complication of cancer treatment, currently has no cure. Lymphedema patients have decreased quality of life and recurrent infections with treatments limited to palliative measures. Accumulating evidence indicates that T cells play a key role in the pathology of lymphedema by promoting tissue fibrosis and inhibiting lymphangiogenesis. Here using mouse models, we show that topical therapy with tacrolimus, an anti-T-cell immunosuppressive drug, is highly effective in preventing lymphedema development and treating established lymphedema. This intervention markedly decreases swelling, T-cell infiltration and tissue fibrosis while significantly increasing formation of lymphatic collaterals with minimal systemic absorption. Animals treated with tacrolimus have markedly improved lymphatic function with increased collecting vessel contraction frequency and decreased dermal backflow. These results have profound implications for lymphedema treatment as topical tacrolimus is FDA-approved for other chronic skin conditions and has an established record of safety and tolerability. Secondary lymphedema is a debilitating disease with no cure. Here the authors show that topical application of an FDA-approved anti-T cell drug tacrolimus potently prevents development and alleviates pathologic changes of established lymphedema in mice, suggesting a new treatment for human patients. Secondary lymphedema (henceforth referred to as lymphedema) is a chronic debilitating disease that in the United States and Western countries occurs most frequently as a complication of cancer treatment. It is estimated that as many as one in three patients who undergo lymph node dissection will go on to develop lymphedema, and conservative estimates suggest that as many as 50,000 new patients are diagnosed annually1Lymphedema is disfiguring and debilitating; patients have chronic swelling of the affected extremity, recurrent infections, limited mobility and decreased quality of lifeFibrosis is a clinical hallmark of lymphedema and recent studies have suggested that lymphedema may represent end stage fibrotic organ failure of the lymphatic system81112+ cells play a crucial role in the regulation of fibrosis in both clinical and animal models of lymphedema. For example, we have found that clinical biopsy specimens of lymphedema and animal models of lymphedema are infiltrated by CD4+ cells, and that the number of these cells correlates with the degree of fibrosis and clinical severity of lymphedema+ cells, than those with early-stage disease. Improvements in clinical symptoms of lymphedema after lymphovenous bypass, a procedure in which obstructed lymphatics are shunted to the venous circulation, is associated with decreased tissue fibrosis and decreased CD4+ cell infiltration+ cell response in lymphedema, similar to other fibroproliferative disorders, is characterized by a mixed Th1/Th2 cell population+ cells (but not other inflammatory cell types including CD8+ cells or macrophages) or inhibition of Th2 differentiation markedly decreases the degree of fibrosis, increases lymphangiogenesis and lymphatic fluid transport, and effectively treats established lymphedema in preclinical mouse models1416+ cells in lymphedematous tissues decrease lymphatic function through multiple mechanisms including induction of structural changes of lymphatic vessels secondary to tissue fibrosis and inhibition of collateral lymphatic vessel formation. Thus, strategies targeting generalized CD4+ inflammatory responses or inhibition of Th2 differentiation may hold clinical promise for treating lymphedema. In addition, because lymphedema is primarily a disease of the skin and subcutaneous soft tissues of the extremities, it may be possible to use topical approaches to accomplish these goals, thereby avoiding systemic complications.More recently, we have shown that CD4Streptomyces tsukubaensis that is well tolerated when used for prevention of transplant rejection and treatment of a variety of autoimmune diseases. It exerts its anti-T-cell properties by binding to FK-506-binding protein 12, thus inhibiting calcineurin, and ultimately decreasing IL-2 expression+ T cells, calcineurin inhibitors have profound CD4+ cell immunosuppressive effects26Tacrolimus is an anti-T-cell agent that is FDA-approved as a topical formulation and used to treat cutaneous inflammatory/fibrotic diseases including atopic dermatitis+ T cells, and their cytokines, play a critical role in lymphedema pathology, the purpose of this study was to evaluate the efficacy of the immunosuppressive drug, tacrolimus, applied topically for the prevention and treatment of lymphedema. Using mouse tail lymphedema and popliteal lymph node dissection (PLND) models, we show that topical tacrolimus potently prevents development of lymphedema after lymphatic injury, as well as alleviates pathologic changes of established lymphedema. Topical tacrolimus has low systemic absorption, but markedly decreases skin CD4+ cell infiltration and fibrosis while increasing formation of lymphatic collaterals. In addition, animals treated with tacrolimus had increased lymphatic collecting vessel-pumping with significantly improved lymphatic function. These findings have important implications for the treatment of lymphedema and represent a paradigm change for treatment since previous experimental attempts have focused exclusively on increasing lymphatic growth and function using exogenous lymphangiogenic growth factors.Considering that CD4To study the effect of topical tacrolimus on lymphedema, we used a well-described mouse tail model of lymphedema2829303128293031−1) remained significantly below the therapeutic immunosuppressive levels achieved with systemic administration  lymphoscintigraphy, a technique in which a radiotracer is injected in the distal tail and uptake by the sacral lymph nodes is measured over time. Decay-adjusted uptake of the sacral lymph nodes in the early treatment animals demonstrated a more than sixfold increase in 99mTc uptake in tacrolimus-treated animals as compared with controls (To assess lymphatic function we performed near-infrared (NIR) lymphangiography with indocyanine green (ICG), which has been described as an effective means of quantifying lymphatic function in humans, pigs and mice by enabling real-time imaging of lymphatic vessels, calculation of packet frequency (or pulsatile flow of lymphatic fluid), and analysis of dermal backflow and dye clearance3738controls . Late tr+ cells  by inflammatory cells  have been described in human patients with lymphedema+ by immunofluorescence) was increased in the tacrolimus-treated group  and vehicle-treated LECs  at Memorial Sloan Kettering Cancer Center. Each experiment was performed using a minimum of six to eight animals, and assays were performed in triplicate. All cell counts were performed by reviewers blinded to the intervention.The studies were performed using adult female (10–14 week old) C57BL/6 J mice  that were maintained in light- and temperature-controlled pathogen-free environments and fed Tail surgery and lymphatic ablation were performed as previously published40−1 intraperitoneally daily. Vehicle control for systemic tacrolimus was the equivalent volume of 10% ethanol, 1% tween 80 in PBS. Cutaneous lymphangiogenesis was assessed using an ear punch wound model as previously reportedA corneal lymphangiogenesis assay was performed as previously reported61Flow cytometry was performed on peripheral blood samples as previously reported4 containing ascomycin as an internal standard. Following a 30-min incubation and centrifugation, 50 μl of the supernatant was injected into the turbulent flow chromatograph system. The analytes were eluted through the column (0.75 ml min−1) with a gradient of water and methanol solutions containing 10 mM ammonium formate and 0.1% formic acid. The analytical run time was 4.5 min. The between-day imprecision of the assay was determined at three concentrations over a period of 10 days. At concentrations of 3.3, 12.6 and 31.9 ng ml−1 the coefficients of variation were of 9.8%, 7.0% and 7.8%, respectively. The assay has a linear range from 0 to 40 ng ml−1.Blood levels of tacrolimus were measured using mass spectrometry in a modification of a previously reported methodTail volumes were calculated using the truncated cone formula as previously reported99mTc) sulfur colloid  was injected in the distal tail. Images were taken using an X-SPECT camera  and region-of-interest analysis was performed to derive decay-adjusted counts in the sacral lymph nodes and to calculate peak and rate of nodal uptake using ASIPro software .Lymphoscintigraphy was performed using our previously published methods−1) ICG  was injected intradermally in the web space of the dorsal hindlimb and visualized using an EVOS EMCCD camera  with a LED light source . Static/video images were obtained using a Zeiss V12 Stereolumar microscope  and lymphatic pumping function was analysed using Fiji software  by identifying a region-of-interest over the dominant collecting vessel of the leg and subtracting the background fluorescent intensity plotted over time.NIR was performed using a modification of previously published resultsImmunohistochemical staining was performed using our published methods+ and CD4+ cell counts were assessed by counting positively stained cells within 50 μm of the most inflamed lymphatic vessel in each quadrant of the leg. Positively stained cells were counted by two blinded reviewers in four randomly selected, × 40 high-power fields in a minimum of four fields per animal. Collagen I deposition was quantified using the Metamorph software (Molecular Devices) in dermal areas of 5 μm cross-sections. This analysis was confirmed using picrosirius red staining and scar index calculation as previously reportedIF staining was performed using AlexaFluor fluorophore-conjugated secondary antibodies . Images were scanned using Mirax imaging software (Carl Zeiss). Perilymphatic CD45Paraffin sections of tail tissues were stained with the Picrosirius Red Staining Kit  according to the manufacturer's instructions. Images were obtained through polarized light on an Axiocam 2 microscope (Carl Zeiss), and the scar index was quantified with the Metamorph software by calculating the ratio of red-orange: green-yellow fibres with higher numbers representing increased scarring.RNA extraction was performed on tail skin using TRIZOL  according the manufacturer's recommendations. RNA quality and quantity was assessed using an Agilent Bio Analyser . The isolated RNA was converted to cDNA using a TaqMan reverse transcriptase kit  and relative expression of gene expression between groups was performed using delta–delta CT PCR analysis and normalizing gene expression using GAPDH RNA amplification as previously describedg for 10 min and the supernatant was collected as our protein sample. Sample protein concentration was determined using the Bradford protocol for protein concentration. Protein concentrations were standardized and enzyme-linked immunosorbant assay was performed to measure inflammatory cytokines TGF-β1, INF-γ, IL-4 and IL-13 (eBioscience) and growth factors VEGF-A and VEGF-C  as per the manufacturer's recommendation, and all experiments were run in duplicate to ensure internal consistency.Inflammatory cytokine analysis was performed on isolated hindlimb protein harvested from mice that underwent PLND for each group. Briefly, hindlimb skin tissue was harvested from mice, ground using a mortar and pestle following flash-freezing using liquid nitrogen, following which protein was extracted using a 100:1:1 mixture of tissue extraction protein reagent , protease inhibitor and phosphatase inhibitor (Sigma-Aldrich). Following extraction, samples were spun using a centrifuge at 16,060Miles assay for vascular permeability was performed as describedIn vitro migration, tube formation and proliferation assays were performed as describedin vitro assays. Briefly, for migration assay confluent LEC monolayer in a six-well dish was scratched with 200 ml pipette tip and supplemented with 100 nM and 1 μM concentrations of tacrolimus and cultured for 24 h. Phase contrast images were acquired at 12 and 24 h, and wound closure area was measured and plotted to represent LEC migration. For tube formation, 1 × 105 LECs suspended in 300 μl of growth factor reduced matrigel (BD Biosciences) and plated in each well of a 24-well dish. Plates were cultured with EGM-MV2 media (PromoCell) supplemented with 100 nM and 1 μM concentrations of tacrolimus. At 24 h, phase contrast images of tubules were taken and analysed for tube length. For proliferation assay, LECs were cultured in chamber slides  with 100 nM and 1 μM concentrations of tacrolimus. After 24 h, cells were fixed and stained for Ki67 (Abcam) and quantified based on confocal image analysis. In another set of experiments after 24 h culture of LECs with desired tacrolimus concentrations, EdU/5-ethynyl-2′-deoxyuridine  was added in culture media for a brief period. Cells were harvested, fixed, permeabilized and stained, and EdU-positive cells were quantified using Flow cytometry . All the experiments were performed in quadruplicates or more, and DMSO was used as vehicle control.P≤0.05, and differences between the two groups was assessed with the two-tailed Student's t-test while multiple analyses were performed using analysis of variance with post hoc tests to compare within groups.A sample size of six to eight animals per group was chosen based on our previous experience with these models and preliminary data. Data were analysed and displayed using the GraphPad Prism software . Values are presented as mean±s.d. unless otherwise noted. Statistical significance was set at All the relevant data are available from authors upon request.How to cite this article: Gardenier, J. C. et al. Topical tacrolimus for the treatment of secondary lymphedema. Nat. Commun.8, 14345 doi: 10.1038/ncomms14345 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures.Lymphatic collecting vessel contractions in animals treated with or without topical tacrolimus 4 weeks after PLND. Time lapse video of NIR lymphangiography in control and tacrolimus treated mice 4 weeks following PLND demonstrating markedly decreased contraction frequency and increased dermal back flow (bottom of left panel) in control mice. This video is shown at 60x normal speed. (n=6/group).Lymphatic collecting vessel contractions in animals treated with or without topical tacrolimus 4 weeks without PLND. Time lapse video of NIR lymphangiography in control and tacrolimus treated mice for 4 weeks without PLND. There was no significant difference in vessel contraction frequency. This video is shown at 60x normal speed. (n=6/group).


Occult primary ovarian insufficiency  is defined as serum AMH level ≤1.1ng/mL in women under age 30. Limited data is available regarding the prevalence of occult POI, the preceding menstrual characteristics and its natural course in otherwise healthy young females. We aimed in this prospective observational study to determine the prevalence of occult POI in young females (< age 30) screened with serum AMH measurement; and analyze the patterns of change in their menstruation at initial assessment and one-year follow-up in relation to the changes in ovarian reserve quantitatively assessed with AMH and AFC. 963 young female college students under age 30 voluntarily participated in this study. 43 of them (4.4%) were diagnosed with occult POI as their AMH levels were ≤ 1.1ng/mL. Thirty-eight (83.4%) of them have regular cycles and denied any menstrual irregularity in the last 12 months. This rate was not statistically different from 7.3% of those with AMH>1.1ng/mL who reported at least one abnormal menstrual cycle in the last year (p = 0.36). Cycle length was significantly shorter in females with AMH ≤ 1.1ng/mL compared to those with AMH>1.1ng/mL . Karyotype, FMR-1 mutation analyses and auto-antibody screening returned normal in all. At one-year follow-up AMH, AFC and mean cycle length were further reduced compared to their values at initial assessment. Now, a greater proportion of the participants with occult POI were menstruating regularly at every 21 days compared to the initial evaluation one year ago . Twenty-five underwent oocyte cryopreservation. These findings underscore the importance of screening young females with AMH for possible occult POI. It also emphasizes that young females with critically diminished ovarian reserve may continue to menstruate regularly without any characteristic menstrual abnormality other than shortening of cycle length. Primary ovarian insufficiency (POI) is a condition in which women under the age of 40 experience oligomenorrhea or amenorrhea for 4 months or more, in association with serum follicle stimulating hormone (FSH) levels in the menopausal range on two occasions obtained at least one month apart according to the most recent ESHRE guidelines , 2. POI Occult form of POI was initially described as the triad of infertility, regular menses and elevated plasma FSH concentrations . There iThe study was approved by the Institutional Review Board of Istanbul Aydin University (IRB#: B.30.2.AYD.0.00.00–480.2/018). Since this study aims to identify young females with occult POI  all students under age 30 were invited to participate in the study using flyers in the main campus of Istanbul Aydin University. There were no pre-selection criteria for inclusion in the study. All participants who were within the required age range were evaluated regardless of their menstrual pattern. There were some exclusion criteria were as follows: the presence of metabolic-endocrine diseases, polycystic ovarian syndrome (PCOS), endometriosis and/or endometriomas, current or past use of oral contraceptive pills (OCP) or other drugs that may affect ovulatory function and hypothalamic-pituitary-ovarian axis (HPO), history of exposure to gonadotoxic chemotherapy regimens and/or radiation for malignant or non-malignant conditions, history of ovarian diseases or surgery, unilateral or partial oophorectomy. A total of 963 young female college students under age 30 voluntarily participated in this study after excluding 18 for OCP use (n = 4), PCOS (n = 12), history of surgical removal of unilateral endometrioma (n = 1), Hodgkin lymphoma survivor previously exposed to cyclophosphamide (n = 1). All participants gave informed consent. They were asked to fill a questionnaire regarding the status of their menstrual cycles, menstrual symptoms, life style, exercise, contraceptive use, personal and family history of endocrine and metabolic diseases, and premature ovarian failure (POF)  and anti-adrenal  antibodies in the blood samples at early follicular phase. Antral follicle counts (AFC) (2-10mm) were determined, and ovarian pathology (such as endometrioma and dermoid cyst) were ruled out using ultrasound exam with Voluson E8  transvaginal or transabdominal probes. Transvaginal ultrasound was not performed in 4 out of 43 (9.3%) participants with occult POI due to their virginity. In these cases, transabdominal approach was performed. The ultrasonographers were blind to the AMH results. Genetic work up was also done for karyotype analysis and pre-mutation (61–199 CGG repeats) and intermediate (41–60 repeats) alleles in the FMR-1 gene. The participants were given a menstrual record chart to keep the records of menstrual cycles until the next visit one year later , any abnormal pattern of bleeding , FSH (Elecsys® FSH) and LH (Elecsys® LH) were used according to manufacturers’s instructions. The lower detection limits for E2, FSH and LH were 5.0 pg/mL, 0.100 mIU/mL, and 0.100 mIU/mL, respectively. All analyses were performed on Cobas® 6000 analyzer series (Roche Diagnostics). AMH levels were determined using Active Mullerian Inhibiting Substance/ Anti-Mullerian Hormone (MIS/AMH)  ELISA kit. The analytical sensitivity of the kit was 0.006 ng/mL. Intra-assay repeatability and the coefficient of variations were given as 4.6% (0.144 ng/mL) and 2.4% (0.843 ng/mL), respectively.Serum samples were collected via venipuncture in the antecubital veins and stored in -80°C until assayed. Electrochemiluminescence immunoassay  kits specific for estradiol  explained this association more optimally than does a linear type regression model   . This isl R2 = 0., p<0.0012 = 0.11, p<0.001). This association was more significant for the participants with occult POI  compared to those without it   of the females diagnosed with occult POI denied any menstrual irregularity in the last 12 months including those with more drastically diminished ovarian reserve (AMH<0.5ng/mL). The remaining five (11.6%) reported mid-cycle spotting (n = 2) and spotting before menstruation (n = 3). This rate was no different from 7.3% (68/920) of those with AMH>1.1ng/mL who reported at least one abnormal non-specific menstrual cycle in the last year (p = 0.36). However, when the cycle length was questioned, it appeared that significantly higher proportion of females with occult POI were complaining of shortening of their cycles in the last twelve months compared to those without occult POI . The cycle length was found to be significantly shorter in the participants with occult POI compared to those without it . Overall p<0.01) . 26 of 4 p<0.01) .2 = 0.19, p = 0.003). Only two participants had elevated FSH (>25 mIU/mL) at early follicular phase with the remaining having normal FSH, LH and E2 levels  were comparable to the first readings  , Fig 3. 2 levels . Anti-thThe third visit took place 12.2±0.3 months later. All participants returned for this visit. Their repeat hormone tests revealed that AMH  and AFC  were significantly declined and FSH was significantly elevated  compared to their corresponding values at initial assessment , Fig 4. We obtained several important findings in this prospective observational study. First, this study confirms one more time that women with critically diminished ovarian reserve may continue to menstruate regularly, emphasizing the fact that we should not rely on regular menses as an indicator of good ovarian reserve. Second, shortening of cycle length precedes menstrual irregularity, making it a more reliable and earlier indicator of diminishing ovarian reserve than any other pattern of menstrual changes in individuals with occult primary ovarian insufficiency (POI). Third, it underscores the importance of screening of young females with possible occult POI with serum AMH measurement as it may develop insidiously in young females with regular menstrual cycle. This is particularly important given that the exact prevalence of occult POI in otherwise healthy young females is largely unknown. It is also unclear if occult POI is precursor of or; distinct from its clinically evident form in terms of prevalence, underlying pathology and natural course. Further, there is no an established method to identify young women with occult POI. Many young women do not regularly visit any health care profession in reproduction especially if they have regular menses and are not interested in childbearing yet. Rather, they are more commonly evaluated by general gynecologists who might not have enough experience and easily overlook low AFC on ultrasound exam that is discordant with patient’s age. Thus, occult POI may remain undiagnosed until it progresses into more advanced stages of low ovarian function and finally become symptomatic as menstrual abnormality and infertility. Even though ESHRE (European Society of Human Reproduction and Embryology) classification of POI did not include AMH as a marker of ovarian reserve, several other studies demonstrated its role in predicting POI in healthy women and Turner’s syndrome patients –16. In tIn contrast to menopause in which permanent cessation of menstruation occurs as a result of depletion of primordial follicle pool, most women with POI may have intermittent and unpredictable ovarian function because the underlying pathology of POI can also be follicle dysfunction in addition to depletion of follicle reserve. The proposed mechanisms of follicle dysfunction in POI include autoimmune lymphocytic oophoritis –19; inapPre-mutations in the gene FMR-1  is well-established cause of primary ovarian insufficiency . There aOur study has several important limitations. It is unclear whether these findings can be extrapolated to a larger population. Transvaginal ultrasound was not performed in 4 out of 43 (9.3%) patients due to their virginity. Even though these ones constitute only a small fraction of all cases it may limit the reliability of AFC measurements. Early follicular phase FSH measurements may have large inter-cycle variability. Long term follow-up is not available and thus fertility outcome and the timing of menopause cannot be assessed. The ovarian reserve and menstrual characteristics of the participants with AMH>1.1ng/ml were not evaluated one year later. Thus, it is unclear how their ovarian reserve and cycle characteristics changed after one year in comparison to their initial assessment and those with occult POI. Another limitation of our study is that cycle length was reported based on recall at the first screening. There may have been recall bias here. At the second screening, cycle length was reported based on menstruation calendar, thus minimizing recall bias and possibly increasing the number of recalled skipped menses. Besides, these women may have been extra alerted as they were selected to be prospectively followed, possibly creating another recall bias. The threshold AMH value to define occult POI is somewhat arbitrary. It remains to be answered how reliably this cutoff level discriminates women with occult form of POI does from those without it. The ESHRE consensus on the definition of poor responders known as the Bologna criteria considered this cutoff value (0.5–1.1ng/mL) in order to predict the poor response of IVF patients to gonadotropin stimulation based on the area under the curve analyses. It was not aimed to predict reproductive life span of females whose serum AMH are below this threshold level. AMH may exhibit assay-specific pre-analytical instability . SeveralThis study underscores the importance of screening young females for possible occult POI as it may develop insidiously without any heralding signs of menstrual abnormality other than shorter cycles. It also appears that occult POI may deteriorate and menstrual abnormalities may begin as early as one year after diagnosis even though it may not fully evolve into clinically evident POI. Declining AMH levels in individuals with occult POI in a relatively short period of time as early as one year as we showed in this study contradicts with the classical description of this clinical entity as a continuum or persistence of impaired ovarian reserve or function. The option of oocyte cryopreservation increased public awareness of ovarian reserve testing in young females who are not currently interested in family building. It may help identify those women with occult POI. However, given that oocyte cryopreservation for social reasons might not be widely available or its regulations by law may change according to the countries, screening of young females with AMH for occult POI might be a valuable tool.S1 TableIt includes the questions to gather information about baseline demographic and menstrual characteristics of the participants along with their personal and family history of diseases and surgeries that may affect ovarian reserve and timing of menopause.(DOCX)Click here for additional data file.S2 TableIt includes the questions to gather information about baseline demographic and menstrual characteristics of the participants along with their personal and family history of diseases and surgeries that may affect ovarian reserve and timing of menopause.(DOCX)Click here for additional data file.S3 TableEvery participant diagnosed with occult primary ovarian insufficiency (POI) was requested to keep the records of their one-year menstrual function using this record chart.(DOCX)Click here for additional data file.S4 TableEvery participant diagnosed with occult primary ovarian insufficiency (POI) was requested to keep the records of their one-year menstrual function using this record chart.(DOCX)Click here for additional data file.S1 Information(SAV)Click here for additional data file.S2 Information(SAV)Click here for additional data file.


The concentration values of direct and indirect biomarkers of ethanol consumption were detected in blood (indirect) or hair (direct) samples from a pool of 125 individuals classified as either chronic (i.e. positive) and non-chronic (i.e. negative) alcohol drinkers. These experimental values formed the dataset under examination . Indirec Specifications TableValue of the data•The data reported here represent a valuable collection of all the common biomarkers of alcohol abuse used worldwide; the distinct populations of chronic and non-chronic alcohol consumers can possibly be used by other researcher to develop further interpretation models.•The Empirical Cross Entropy plots provide a novel way to look at the effectiveness of alcohol biomarkers that other researcher may use for comparison with more traditional data representations.•The detailed data report allows a clear comparison between univariate, multivariate and Bayesian approaches, where the latter is suggested as a benchmark for further developments.•The mathematical background reported in the “materials and methods” section allows other researcher to transpose the offered approach to different applications.1Data relative to the population of 125 individuals monitored, previously classified as either chronic (i.e. positive) and non-chronic (i.e. negative) alcohol drinker, are available in 2® auto-analyzer . MCV was measured with an ADVIA® 2120 Hematology auto-analyzer . The %CDT was determined by the HPLC reagent kit purchased by BioRad® . FAEEs were detected by HS–SPME–GC/MS analysis and a MultiPurpose Sampler Flex A05-FLX-0001  equipped with a 65 μm StableflexTM polydimethylsiloxane/divinylbenzene fiber (PDMS/DVB) from Supelco  was used in combination with a 6890N GC 5975-inert MSD . EtG concentrations were monitored by UHPLC–MS/MS analysis and a Shimadzu Nexera UHPLC system  interfaced to an AB Sciex API 5500 triple quadrupole mass spectrometer  was employed.Ethical approval for the study was granted by the Ethical Committee of the Azienda Ospedaliero-Universitaria San Luigi Gonzaga of Orbassano (Protocol Number 0012756). Serum activities of AST, ALT and GGT were measured by means of colorimetric assays with a Roche-Cobas Integra 800Descriptions about the analytical methodologies utilized to detect both the direct and the indirect biomarkers are available in 10x) was applied on the analyzed data. Before calculating the different LR models, all the variables were autoscaled and equal prior probabilities were utilized. LR evaluations (briefly represented by this formula LR=Pr(E|H1)/Pr(E|H2)) involved two mutually exclusive hypotheses  and a reference population was used to build the model, representing the experimental evidence (E). The ECE plots relative to indirect biomarkers detected in blood samples are reported in Base 10 logarithm transformation  and a shorter list of variables  are shown in ECE plots relative to the sum of the four FAEEs and EtG are reported in Multivariate approaches were also performed on the collected data simultaneously; Principal Components Analysis The formulas employed, together with the description of ECE plots, are reported in


Chronic 1% metformin treatment is nephrotoxic in mice, but this dose may nonetheless confer health benefits if given intermittently rather than continuously. Here, we examined the effects of 1% metformin given every-other week (EOW) or two consecutive weeks per month (2WM) on survival of 2-year-old male mice fed standard chow. EOW and 2WM mice had comparable life span compared with control mice. A significant reduction in body weight within the first few weeks of metformin treatment was observed without impact on food consumption and energy expenditure. Moreover, there were differences in the action of metformin on metabolic markers between the EOW and 2WM groups, with EOW metformin conferring greater benefits. Age-associated kidney lesions became more pronounced with metformin, although without pathological consequences. In the liver, metformin treatment led to an overall reduction in steatosis and was accompanied by distinct transcriptomic and metabolomic signatures in response to EOW versus 2WM regimens. Thus, the absence of adverse outcomes associated with chronic, intermittent use of 1% metformin in old mice has clinical translatability into the biology of aging in humans. Chronic daily exposure to a high dose of metformin  shortens lifespan of non-diabetic mice, although in the short term this treatment confers a similar pattern of gene expression and phenotypes consistent with the benefits of caloric restriction. A team of researchers led by Rafael de Cabo at the National Institute on Aging, NIH tested whether a strategy of intermittent 1% metformin treatment in old mice alters the course of aging and avoids toxicity. They found that when metformin was given every-other-week, it significantly improved insulin sensitivity and reduced age-associated liver lesions without having a negative impact on maximum lifespan in male mice. The absence of adverse outcomes associated with the use of 1% metformin in old mice has clinical translatability into the biology of aging in humans. Metformin has an established safety record and its current approved indication is to improve glycemic control in adults and children with type 2 diabetes mellitus along with diet and exercise.8 and mitochondrial glycerophosphate dehydrogenase (GPD2).9 The ensuing perturbations in cellular energy status and redox homeostasis result in activation of the energy sensor AMP-activated protein kinase (AMPK). AMPK-independent actions of metformin have been also described,14 which include the reduction in mTOR/p70S6K activity resulting from Rag GTPase inhibition.10 The anti-gluconeogenic action of metformin is preserved in liver-specific AMPK knockout mice.13 Moreover, this biguanide confers protection against certain cancers by inhibiting mTOR/p70S6K activity in an AMPK-independent manner,15 and reduces the tumorigenicity of induced pluripotent stem cells while maintaining their pluripotency.16 Impaired cellular senescence and accumulation of DNA damage contribute to aging17 and metformin has been shown to trigger an immune-mediated clearance of senescent cells by activating an ATM-dependent DNA damage response.18 The ability of metformin to lower the incidence of diabetes-associated cardiovascular events and risk of cancer without interfering with stem cell pluripotency may have important implications for a number of age-related diseases that affect lifespan (for recent review19).The primary target of metformin appears to be the mitochondrion through inhibition of complex I of the respiratory chain21 Our previous study showed that diet supplementation with a low dose of metformin (0.1% w/w) improves healthspan and extends lifespan when treatment was started in mice at middle age.2 However, long-term exposure to a high dose of metformin  is nephrotoxic and leads to early mortality in mice even though global gene expression profile appears to be closer to CR when given only for a short period of time.2 Nevertheless, there has been a recent flurry of interest in the use of metformin in clinical translational research on aging. In that regard, a new clinical trial has been set up to investigate the health benefits of chronic metformin treatment and determine whether it can induce dietary restriction-like state in humans . A second clinical study, which is known as the “Targeting Aging with Metformin” study, aims at assessing the potential of a pharmacological intervention in delaying age-associated diseases beyond metformin’s isolated impact on diabetes.22 A similar paradigm is currently being tested in the pilot study known as “Metformin in Longevity Study” . In the latter trial, investigators will search for gene expression changes in adults 60 years and older to provide initial clues on how and whether an older molecular and genomic signature can be reverted to a younger one. These studies will take years before providing meaningful information on the healthspan and lifespan potential of metformin in humans.Metformin is a calorie restriction (CR) mimetic, with several of its metabolic actions, particularly those related to glucose metabolism, resembling to some extent those of CR.2 The results indicate that intermittent metformin treatment did not lead to early mortality and that EOW metformin improves some metabolic markers of health without promoting lifespan extension when treatment is initiated in late-life in mice. The mechanisms potentially underlying these effects are discussed.In order to provide timely insights into the potential pro-longevity benefits of this drug, we set out to examine the effect of intermittent treatment with 1% metformin given either every-other-week (EOW) or two consecutive weeks per month (2WM) in 2-year-old male mice. We chose EOW and 2WM in order to potentially reduce the impact and incidence of nephrotoxicity observed in our previous study, while conferring CR-like health benefits.2 Here, we asked whether intermittent feeding with 1% metformin in the diet could confer protection in the absence of toxicity in a cohort of 2-year-old male mice.Daily exposure to low dose of metformin (0.1% w/w in diet) confers healthspan benefits and extends lifespan when given to middle-age male mice while a higher dose (1% w/w) was found to be toxic.n = ~65 mice per experimental group × 3 groups). The collection of blood samples and measures of physiological/biochemical parameters was carried out as depicted in Fig. 2 EOW and 2WM mice consumed about 1 g metformin kg−1 body weight per day, which translates to about 80 mg metformin kg−1 body weight in humans,23 a dosage that is more than twice the maximum dose of metformin that patients receive (3 × 850 mg per day). Weight trajectory of the three groups is shown in Fig. Male C57BL/6 mice were fed a standard AIN-93G diet (SD) supplemented with 1% metformin EOW or for 2 consecutive weeks each month (2WM) for the remainder of their lives (2 production vs O2 consumption) in old EOW and 2WM mice while on metformin was similar to that of SD mice  to determine circulating glucose and lactate levels after a 6-h fast. While being unaffected in EOW mice, the fasting blood glucose levels were significantly lower in 2WM mice at week-14, representing the second consecutive week on metformin Fig. . Increasods Fig. . An addiods Fig. . Glycateods Fig. . EOW micods Fig.  that coiods Fig.  despite ods Fig. . Comparaods Fig. . Althoug27 Histochemical analyses were carried out on frozen-fixed liver tissues to determine the effects of intermittent metformin treatment on hepatic steatosis, ballooning, inflammation, and glycogen deposition  of fixed liver sections was performed to assess the integrity of the hepatic sinusoidal endothelium in mice subjected to intermittent metformin treatment  and Saa4 whose transcript encodes the inflammation-inducible serum amyloid A4 protein  showed a clear effect of metformin treatment on liver transcriptome Fig. . To undeein Fig. . Lpin1 eZ-score of −5.604. Positive and negative scores reveal the number of standard deviations the aggregated Z-score of a given GO Term (or pathway) within a pairwise comparison  is either above or below the mean of all pathways .33 As shown in Fig. Srd5a1, a gene implicated in the conversion of testosterone into the more potent androgen, dihydrotestosterone, and Dio1, which converts thyroxine to the bioactive thyroid hormone, whereas both genes were unaffected in 2WM livers. Both fasting and metformin treatment stimulate hepatic Igfbp-2 transcription,35 which is known to negatively regulate GH signaling. The expression of Igfbp2, whose transcript participates in the reduction in the bioavailability of IGFs, was upregulated in EOW, but not 2WM livers , was also found in EOW and CR livers  .cs2 Fig. . MoreoveFdps, Hmcgs1, and Mvd, which are primarily involved in cholesterol and sterol biosynthesis.A binary representation of the 31 significantly altered genes shared between CR40, EOW, and 2WM indicated a subset of transcripts with opposite direction of expression between intermittent metformin treatment and CR Fig. : These iP ≤ 0.05) were merged in two-way Venn diagrams as a mean to identify unique and overlapping ones  , while other molecules such as aspartate, citrate and isocitrate were increased , this work illustrates the complexity of metformin’s physiological effects and underscores the need for a comprehensive assessment of the interaction that exists between metabolic dysfunction and aging phenotypic outcomes.Although the use of metformin as a pro-longevity strategy has been proposed when started early in life,37 reduction in the rate of small intestinal glucose absorption to enhance postprandrial secretion of glucagon-like peptide-1,38 targeting of central carbon metabolism,39 anti-cancer protection,40 and the regulation of microRNA expression and cellular senescence.41 The present study demonstrates significant improvement in insulin sensitivity in non-fasted old mice subjected to intermittent metformin treatment for 17 weeks. This was evidenced by lower circulating insulin levels and HOMA-IR index, despite maintenance of blood glucose levels within the range seen with SD mice. This metformin protocol also reduced fasting plasma glucose and improved insulin sensitivity without translating into lifespan extension, even though limiting basal insulin levels in old mice has been recently reported to exert a positive effect on lifespan.42 We observed lower circulating leptin levels in the metformin-treated mice than in SD controls despite similar lean-to-fat ratio, supporting the idea that this drug is acting as a leptin sensitizer by increasing hepatic and hypothalamic leptin receptor expression.44In addition to its involvement in lowering hepatic glucose production, metformin has recently been implicated as a direct regulator of metabolism, with actions including activation of a neuronal-mediated gut–brain–liver pathway,The rapid weight loss early in the metformin treatment could not be explained by the decrease in food intake or increase in foraging activity, but instead may have been the product of heightened rates of metabolic processes. Examination of the 24-month-old mice in the CLAMS monitoring system revealed the inability of metformin at promoting mitochondrial fatty acid oxidation during light cycle. Moreover, the removal of the drug during the intermittent metformin treatment was accompanied by a shift in carbohydrate utilization in both the light and dark cycles of EOW mice , with a similar trend observed in the 2WM group. The greater reliance on sugar metabolism during the off-metformin period may be reflected by falling circulating lactate levels due to increased production of pyruvate and its ultimate conversion to ATP via aerobic metabolism through complex I of the respiratory chain.13C in mice on high-fat diet reports on the increased conversion of labeled glucose to lactate-3-13C in the intestinal wall due to inhibition of complex I of the mitochondrial respiratory chain45 by metformin, and release of lactate-3-13C into the portal vein with subsequent generation of doubly labeled glucose-1,6-13C molecules in the liver via gluconeogenesis.46 The authors propose that this “futile” intestinal–zhepatic cycle is very energy consuming and could explain some of the reduced weight gain seen in HFD-fed mice on metformin. We reasoned that a similar mechanism could be implicated in the initial weight loss observed in old mice enrolled in the intermittent metformin program.The expression of hepatic genes involved in cholesterol biosynthesis, gluconeogenesis, and ribosome/translation were upregulated following chronic intermittent treatment of old mice with metformin for 17 weeks. Even though metformin has been shown to inhibit hepatic glucose production, a recent study using oral administration of glucose-1-47 It follows that major impact on hepatic lipid and glucose metabolism and energy production is expected when comparing young and old animals. Indeed, our preliminary data clearly showed that the daily cycling of energy expenditure over a 48-h period is markedly dampened with age, with RER values significantly higher during the light phase in 24-mo-old C57Bl/6 mice compared to 5-mo-old animals. These results are consistent with an age-associated substrate preference toward carbohydrate utilization for energy demand at the expense of fatty acid β-oxidation.There are alterations in hepatic AMPK activity during normal aging in mice.48 The integrity of the liver sieve is essential for the maintenance of insulin, glucose, and lipid metabolism and clearance of ingested and gut microbiota-derived metabolites.49 Aging predisposes the liver to chronic low-grade inflammation and oxidative damage that can contribute to the development of fibrosis and non-alcoholic fatty liver disease, which can evolve into non-alcoholic steatohepatitis in the absence of diet-induced obesity and type 2 diabetes.51 A recent study reported that the age-dependent increase in hepatic steatosis relates to cellular senescence.52 The ability of metformin to decrease cellular senescence and lower the abundance of inflammatory cytokines in several senescence models41 is in agreement with our findings that showed significant reduction in hepatic steatosis and liver injury in EOW mice. The failure to maintain homeostasis and adapt to environmental changes drives the pathogenesis of age-related disease of the liver. It is possible that some of the protection against hepatic inflammation in the aged EOW mice on metformin relates to the modulation of the gut microbiota through activation of the immune system.53 Clearly, more work is needed to validate this hypothesis.Because behavioral and motor coordination tests as well as the metabolic panel blood analyses were carried out at a single time point  instead of longitudinally, this study does not provide sufficient information to conclude that metformin promotes healthspan in aged mice. Despite the limitation of the study, several positive outcomes were observed in the liver of EOW mice, notably by histology and SEM, including improvement in the age-related changes in the porosity of the liver sieve.55 and it typically emerges during meal absorption. This intestinal effect of metformin increases anaerobic metabolism of glucose to lactate and its utilization in the periphery. These observations are consistent with our basic serum metabolic panel showing the significant increase in circulating lactate in non-fasting EOW mice. At the time of sacrifice, the liver metabolome of EOW mice on metformin revealed a significant reduction in lactate, which may be the result of its increased conversion to pyruvate and cycling back to glucose via gluconeogenesis.46 Taken as a whole, these findings offer a promising use of metformin in combatting ailments of aging, although gastrointestinal intolerance, lactic acidosis, and kidney failure have contributed to the adverse side-effects associated with metformin use.56 Based on our results, it should be emphasized the importance of optimizing the schedule of metformin administration in order to gain the most health benefits.Metformin-associated lactic acidosis is a known adverse side-effect of metformin therapy, which can be injurious in patients with renal dysfunction. Preclinical and clinical evidences show that the intestine appears to be an important source of metformin-induced lactate productionAngptl4 expression in EOW livers is consistent with earlier reports showing the contribution of this liver-expressed serum hormone in the regulation of glucose homeostasis, lipid metabolism, and insulin sensitivity.57 Like metformin, long-term CR and exercise increase plasma ANGPTL4 levels in humans58 whereas decreased expression of Angptl4 has been associated with type 2 diabetes.57 Lipin-1 plays a central role in mTORC1-dependent regulation of lipogenesis by SREBP159 and we found that Lpin1 mRNA levels were significantly upregulated in EOW and 2WM livers together with an upregulation in pathways related to ribosome/translation. To what extent does the upregulation of genes encoding cholesterol and sterol biosynthetic enzymes and that of key proteins implicated in translation regulation antagonizes the pro-longevity effect of metformin remains to be determined. Noteworthingly, increases in lipoprotein cholesterol concentrations are associated with coronary atherosclerosis and shortened lifespan.60Focusing on the molecular underpinnings of metformin action, transcriptomic and metabolomics analyses have been performed on liver tissue extracts from 2-year-old mice fed SD and mice on intermittent metformin protocol for 17 weeks. Microarray analysis illustrated the fact that EOW treatment impacted hepatic expression of several metabolically-relevant genes, including those encoding the transcriptional activator SREBF1—required for lipid homeostasis, the transcriptional coactivator PGC-1α—implicated in the regulation of genes involved in energy metabolism, and SOCS2—a negative regulator in the GH/IGF-1 signaling pathway. The increase in 32 The analysis suggests great similarity in the gene expression profiles between CR and EOW, but not 2WM  and significant elevation in plasma 2-hydroxybutanoic acid in old EOW mice was consistent with active transsulfuration pathway.Tryptophan has been recently identified as a putative biomarker for type 2 diabetes riskity Fig.  with ass71 Herein, EOW livers had significant reduction in palmitoleic acid, which is consistent with improved insulin sensitivity and lower hepatic triglyceride content.Hepatic conversion of glucose into fatty acids generates palmitoleic acid, a metabolite implicated in the regulation and pathophysiology of insulin action and glucose metabolism. A study on the relationship between palmitoleic acid and metabolic risk revealed that circulating palmitoleate is positively associated with greater adiposity, higher triglycerides, and greater insulin resistance in humans.To conclude, the metabolic effects of metformin EOW when initiated in late-life were associated with an overall improvement on health without an extension in lifespan as compared to control mice. A combination of confounding factors such as adiposity, energy overconsumption (ad libitum feeding) and related weight gain, mitochondrial dysfunction, and incidence of cancer could explain why metformin was unable to counterbalance these adverse life-shortening events despite exhibiting favorable markers of lower metabolic risk. Thus, additional longitudinal studies will need to carefully assess the effects of intermittent metformin treatment introduced at young to middle-age and how gender and rodent genetic background may modify or confound metformin’s effectiveness at improving healthspan, and possibly lifespan. Targeting lifespan extension is a very complex endeavor especially when a given intervention  is provided in late-life once evolutionarily conserved molecular pathways are already impaired, with genome stability loss, DNA repair defects, and chronic oxidative stress to name of few. The fact that metformin did not generate adverse outcomes in very old mice is a reassuring safety data possibly for humans that start metformin earlier in life and want to continue it into old age. Further focus on classical hallmarks of aging should provide new insights into whether/how improving healthspan can lead to lifespan extension.Full details are provided in the n = 68) until their death while the other groups of mice were fed SD plus 1% metformin following different on-and-off feeding protocols. Feedings protocol consist of every other week  and two consecutive weeks each month  with AIN-93G plus 1% metformin until their death. Sample size estimate to ensure adequate power to detect change in maximum lifespan was carried out based on our previous published work. Pure metformin was obtained from Farmhispania  and mixed to homogeneity during manufacturing of the diets . The chow was produced every 3 months during the length of the study, never was permitted to exceed 50 °C and was kept away from light whenever possible to ensure the stability of metformin . The mice were on a light:dark 12:12-h schedule and maintained between 20–22 °C with 30–70% relative humidity according to animal protocols and NIH guidelines. Food intake and body weight were measured on a biweekly basis for the duration of the study for control group and weekly for metformin fed mice. Body temperature was biweekly measured through an implantable temperature transponder system . The transponders were implanted under the skin on the back of the animals between the shoulder blades using the provided instrument. The animals were allowed to heal and recover for a minimum of one week after the implantation before measurements were commenced. Survival curves were plotted using the Kaplan–Meier method, which includes all available animals at each time point. The criteria for euthanasia was based on an independent assessment by a veterinarian, according to AAALAC guidelines and only cases, where the condition of the animal was considered incompatible with continued survival, are represented in the curves. Animals removed at sacrifice for experimental procedures were considered as censored deaths.Animal procedures, housing and diets were in accordance with the guidelines issued by the Intramural Research Program of the National Institutes of Health protocol number 444TGB2016. Male C57BL/6 mice at 104 weeks of age were purchased from the National Institute on Aging Aged Rodent Colony from Charles River. The C57BL/6 mice maintained on a standard purified mouse diet (AIN-93G) for a month were randomly distributed to avoid differences in starting body weight average between the 3 experimental groups. Control group was kept on standard diet  after 16 weeks of treatment.2 Movement  was also monitored with beams that software transforms into counts of beam breaks by the mouse.After 16 weeks of metformin treatment, mouse metabolic rate was assessed by indirect calorimetry in open-circuit Oxymax chambers with CLAMS (Columbus Instruments) as previously described.A detailed explanation of all physical performance tests performed is described in the n = 8–10) on the Friday of week 13, 14, 15, and 16 of treatment. Glucose and lactate concentrations in blood were measured by tail venipuncture with the Blood Glucose Monitoring System Breeze 2  and Lactate plus Meter , respectively.Because diet was changed every week for EOW mice and every 2 consecutive weeks for 2WM mice, lactate and glucose levels were measured in 6-h fasted animals (n = 10) and mice on EOW and 2WM metformin (n = 6 each) were euthanized for experimental procedures. Glucose and lactate were measured from a drop of tail blood, with lactate measured from the first drop. Mice were anesthetized with ketamine–xylazine , and then blood and various tissues were collected as summarized in After 17 weeks of treatment, a subgroup of non-fasted SD mice , and insulin levels were determined according to the manufacturer’s protocol .An aliquot of whole blood was mixed with EDTA and used for the direct determination of HbA1c with an enzymatic kit . Coagulated whole blood was centrifuged at 12000×Full methodological details for liver and kidney histology, and the determination of fenestrations in the liver sinusoidal endothelium by SEM can be found in Z-normalization, as described elsewhere.73 PCA was performed on the normalized Z-scores of all of the detectable probes in the samples using DIANE 6.0 software, available from: (http://www.grc.nia.nih.gov/branches/rrb/dna/ diane_software.pdf). Significant genes were selected by the z-test < 0.05, false discovery rate < 0.30, as well as z-ratio > 1.5 in both directions and ANOVA p value < 0.05. Methodological details can be found in the Raw data were subjected to Details can be found in the 32 Methodological details can be found in the Metabolomics analysis on mouse liver extracts and serum was performed by the UC Davis West Coast Metabolomics Center as previously described.26 Quantification was performed by volume densitometry using ImageJ software.The measure of hydrogen sulfide was carried out in liver extracts following the methodological details previously described.n = 12) and liver homogenates (n = 14) was performed using a LC-MS/MS method developed at SRI International , as described in −1 in serum and 186 ng g−1 tissue in liver.Method to measure metformin levels in mouse serum . In all experiments, results are represented as the mean ± SEM, with Microarray data have been deposited in the Gene Expression Omnibus database under accession code GSE97074. All relevant data that support the findings of this study are included in this published article and its , or are Supplemental Material


Higher serum BDNF was an independent factor for greater reduction in pulse pressure . Using a receiver operating characteristic curve analysis, serum BDNF >20.6 ng/mL predicted a pulse pressure reduction after a one-hour rest . In conclusion, higher serum BDNF predicted greater recovery of pulse pressure after a one-hour rest in female nurses after night shift work.Night shift work is associated with cardiovascular disease and central nervous system disorders in female nurses. Brain-derived neurotrophic factor (BDNF) exerts protective effects on neural and endothelial functions. This study examined the association between serum BDNF levels and pulse pressure after rest in female nurses working night shifts. In this study, blood samples were collected for BDNF measurement after a night shift when nurses had been working night shifts for three continuous weeks. Blood pressure was assessed before and after a one-hour morning rest within a week of resuming the night shift after one month without any night shift work. The pulse pressure of nurses  was significantly reduced  after rest, and serum BDNF were significantly and inversely correlated with pulse pressure changes ( According to the data from 39 hospitals in Taiwan, approximately 40% of female nurses had performed night shifts for more than 10% of their total work time2. In other reports from the NHS I, night shift work was significantly associated with coronary heart disease and ischemic stroke in female nurses4. Night shift work was recently reported to be significantly associated with all-cause mortality and cardiovascular mortality in female nurses who were followed for 22 years5.Night shift work is inevitable for nurses caring for inpatients. Pooled data from the Nurses’ Health Study (NHS) I and NHS II show that approximately 60% of female nurses had experienced night shift work7. In one mouse study, the disturbance of circadian rhythms induced by exposure to light at night caused a depressive response8. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays important roles in protecting neurons and synaptic activity9. A reduction in circulating BDNF level is associated with depressive symptoms12, and serum BDNF levels can reflect an improvement in depressive symptoms after lifestyle interventions14 or pharmacological treatments16.Rotating shift work is also associated with the central nervous system disorders17. In one recent cross-sectional study, lower circulating BDNF concentrations were reported to be associated with coronary artery disease18; in a prospective study, a lower BDNF concentration was reported to predict coronary heart disease and higher all-cause mortality19. Therefore, insufficient BDNF levels may play an important role in linking central nervous system disorders and arterial stiffness20.Notably, a low serum BDNF level is associated with forearm vascular resistance21, and a higher pulse pressure predicts a higher cardiovascular mortality rate23. According to studies that stratified by age, pulse pressure is a more powerful predictor of cardiovascular mortality in young subjects than in older subjects25. Furthermore, long-term reduction in pulse pressure is significantly associated with survival26. A short-term reduction in pulse pressure from peak exercise to rest is predictive of survival in subjects with normal myocardial perfusion imaging27, or after hemodialysis in patients with end-stage renal disease28.Pulse pressure, the difference between systolic and diastolic blood pressures, can reflect the severity of arterial stiffening30. According to Mathiassen et al.31 a delayed recovery of blood pressure increased by physical work was observed after a one-hour rest period. In the present study, we examined the relationship between serum BDNF levels and changes in pulse pressure after a one-hour rest period in female nurses working night shifts.Based on psychological stress, delayed recovery of systolic blood pressure after 45 minutes might predict the carotid intima-media thickness and hypertensionThis prospective observational study was conducted at Taichung Veterans General Hospital, Taiwan. The included subjects were female nursing staff members aged ≥20 years who worked in wards with a monthly rotating shift pattern. The exclusion criteria were (1) working night shifts at the time of the screening, (2) known diabetes mellitus, (3) established cardiovascular diseases, (4) current severe infection or inflammation, (5) a history of malignancy, (6) a history of psychological or mood disorder, (7) a history of substance or alcohol abuse, and (8) post-menopause or pregnancy. The study complied with the Declaration of Helsinki. All experimental protocols were approved by the Institutional Review Board of Taichung Veterans General Hospital and the written informed consent was obtained from all subjects. The methods were carried out in accordance with the approved guidelines.The aim of this study was to determine whether serum BDNF levels in female nurses who had worked a stable night shift predicted pulse pressure after a one-hour rest period when they rotated to the next night shift. The study subjects were screened when they were just before they rotated to night shift work for the following month. During the first night shift month, a three-week observation period confirmed that the subjects were performing their night shifts in a stable pattern. In the fourth week, the subjects were scheduled for a morning appointment after overnight fasting. Blood samples were collected to measure fasting glucose, insulin, lipid profile, serum creatinine, C-reactive protein (CRP) and BDNF levels after anthropometric measurements were obtained.After the first night shift month, the subjects underwent a wash-out period during which they did not perform night shift work for a month. The participants were scheduled for another morning visit during the first week of their rotation to a second month of night shift work after the one-month wash-out period. During this visit, blood pressure was measured at the right brachial artery, and the mean of two separate measurements with a interval of 1 min was recorded after the subjects had rested in a lying position for 10 minutes in the morning following the end of the night shift. Blood samples were not collected during this visit to avoid potential impacts of any invasive procedures on the blood pressure measurements. Then, the subjects were asked to take a nap on the same bed for one hour. After the nap, blood pressure was measured again.32. The estimated glomerular filtration rate (eGFR) was calculated as 186 × [serum creatinine (mg/dL)]−1.154 × [age (years)]−0.203 × 0.742 based on the Modification of Diet in Renal Disease (MDRD) equation33.Glucose levels were determined using an oxidase‒peroxidase method , and insulin levels were determined using a commercial immunoassay . Serum levels of creatinine and lipids were measured using commercial kits , and CRP levels were measured using an immunochemical assay with purified Duck IgY (∆Fc) antibodies . Serum human BDNF levels was measured using an enzyme immunoassay method . The intra‒ and inter‒ assay coefficients of variation for BDNF measurements were 4.1% and 9.0%, respectively. The sensitivity of the BDNF measurement was 0.02 ng/mL. The formula [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5 was used to calculate the homeostasis model assessment for insulin resistance (HOMA-IR) indext-test was used to detect significant differences between two groups. A paired t-test was used to detect significant differences in blood pressure before and after the one-hour rest. The correlation between serum BDNF levels and changes in the pulse pressure was determined using Pearson’s correlation coefficient. A multivariate linear regression analysis was used to assess the association between the changes in pulse pressure and serum BDNF levels. Statistical analyses were performed using SPSS version 22.0 software .Continuous variables are presented as the means ± standard deviations (SD). An independent samples A total of 53 female nurses were screened, of whom 48 completed the study assessments Fig. . Table 1According to the changes in pulse pressure after a one-hour rest, we divided the subjects into a pulse-pressure (P-P) reduction group n = 30) and a no P-P reduction group (n = 18). Table 0 and a nBased on the receiver operating characteristic (ROC) curve analysis, a cut-off value of 20.6 ng/mL for serum BDNF levels provided a sensitivity of 66.7% and specificity of 77.8% for differentiating a reduction in pulse pressure after a one-hour rest in nurses performing night shift work Fig. .Figure 434. Widening pulse pressure, resulting from a surge of systolic pressure against a non-compromised arterial wall followed by a low diastolic pressure, reflects the severity of arterial stiffness35. Arterial stiffness may involve not only the extracellular destruction of the vessel wall37, but also dysfunction of vascular smooth muscle cells38. Tropomyosin-related kinase receptor B (TrKB), a high-affinity receptor for BDNF, is expressed in the vascular smooth muscles40. Inactivation of the BDNF-TrkB signaling pathway in vascular smooth muscle cells may impair coronary vascular integrity and function41. Following treatment with empagliflozin, a sodium glucose co-transporter 2 inhibitor, higher BDNF expression was associated with the reduction of coronary arterial fibrosis in a diabetic mouse model42.The main finding in our study was that higher levels of serum BDNF levels in female nurses during night shift work predicted a greater reduction in pulse pressure after a short morning rest when rotating back to night shift work. Arterial stiffening describes the loss of the arterial wall’s elastic capacity to buffer the blood flow from ventricular ejection43. Increased arterial stiffness with decreased vascular activity, estimated using the reactive hyperemia index, has been found in subjects currently performing night shift work when compared with those not currently working night shifts44. Although the high blood pressure experienced during the work period normally decreases after a rest period, a delayed recovery of the high blood pressure during rest periods is more common in female nurses working night shifts than in those working day shifts45. High blood pressure may be the result of circadian disruption when rotating to night shift work. Kubo et al.46 also reported a reduction in the coronary flow reserve of the left anterior descending artery in female nurses the morning after working a night shift.Night shift work interrupts the circadian rhythm and may impair vascular functions controlled by autonomic activity47; however, the change in circulating BDNF was not reported. In the present study, coffee-consumption frequency did not significantly affect the serum BDNF (21.5 ± 5.3 ng/mL in the subjects who consumed coffee <1 time per month vs. 21.7 ± 7.5 ng/mL in the subjects who consumed coffee ≥1 time per month). Although the intake of a single-dose of coffee fruit extract was also reported to increase the circulating BDNF levels of healthy subjects within 120 min48, serum BDNF was assessed in a fasting state in the present study. This may explain why coffee consumption was not associated with serum BDNF levels in the present study. In contrast, it has been reported that arterial stiffness might be induced by either acute or chronic coffee consumption51. Souza et al.52 also reported that a single dose of coffee might increase post-exercise systolic blood pressure. The impact of coffee consumption on pulse pressure recovery after rest following a night shift should be further investigated.Regular coffee consumption increases BDNF expression in the in the mouse hippocampus53, the impact of shift work on mental health has been reported to differ between genders54. Therefore, our findings in female nurses cannot be applied men. In addition, our findings cannot be applied to postmenopausal women since a lower circulating BDNF concentration has been observed in postmenopausal females than in young females56. Second, menstrual cycle information was not recorded for analysis in the present study. An increase in circulating BDNF during the luteal phase compared with the follicular phase has been reported in woman with normal menstrual cycles57. Additionally, Dunne et al.58 reported lower blood pressure during the luteal phase than during the in follicular phase, and Mills et al.59 reported a difference in blood-pressure recovery after stress between the luteal phase and follicular phase. Therefore, the menstrual cycle could be a confounding factor in studies of young females. Third, we did not measure the serum BDNF levels when blood pressure was assessed because of the relationship between BDNF and vascular functions consistently reported in previous studies62. Fourth, the change in diastolic blood pressure was not statistically significant after rest. We did not further examine the underlying causes of differences in recovery between systolic and diastolic blood pressures. Finally, we did not explore the mechanism linking BDNF and pulse-pressure recovery after night shift work; moreover, we did not assess the long-term cardiovascular outcomes in subjects with high pulse pressures after a one-hour rest period.We collected serum BDNF data in the morning after a night shift when the participants had worked the night shift for three weeks, and we measured the morning blood pressure after a night shift in female nurses who had rotated to a new series of night shifts within one week of the rotation. We found that high morning serum BDNF levels during stable night shift work could predict the quick recovery of a widening pulse pressure after a one-hour rest in the female nurses who were beginning a new night-shift schedule. However, the present study had certain limitations. First, the study only included young female nurses. Despite the delayed recovery of blood pressure in men performing night shift workIn conclusion, the serum BDNF concentration measured during a stable period of night shift work predicted a reduction in pulse pressure in female nurses after one hour of rest when they were rotating to a new period of night shifts. These findings may indicate a possible modulatory effect of circulating BDNF on the recovery of pulse pressure in female nurses after they have worked rotating night shifts. Further studies are necessary to investigate the underlying mechanisms linking serum BDNF during night shift work to the long-term cardiovascular prognosis.


MYH11 gene affecting the C-terminal coiled-coil region of the smooth muscle myosin heavy chain, a contractile protein of smooth muscle cells (SMC), have been described to cause thoracic aortic aneurysm or aortic dissection (TAAD) and patent ductus arteriosus (PDA).1 Herein we expand the phenotype associated with MYH11 mutations to include moyamoya-like cerebrovascular disease.Heterozygous mutations in the links.lww.com/WNL/A36). Brain MRI revealed an acute left anterior and middle cerebral artery territory (ACA/MCA) infarct and prior infarction in the right ACA/MCA territory (figure e-1). The clinical correlate of the latter was an episode of reluctance to use the left hand. Catheter angiography showed bilateral stenosis of the terminal internal carotid artery (TICA)/middle cerebral artery with a collateralization pattern consistent with moyamoya arteriopathy  revealed a novel heterozygous missense mutation in MYH11 gene NM_002474:c.4604G>A (p.R1535Q) confirmed with Sanger sequencing .2 The majority of these conditions are characterized by prominent thoracic aorta involvement.1–3 Recently, however, specific mutations, for instance heterozygous missense mutations in ACTA2 disrupting Arg179, have been shown to associate mainly with cerebrovascular disease.4 We now also expand the phenotype associated with heterozygous mutations in MYH11 to include an occlusive cerebral arteriopathy. We would suggest that screening for cerebrovascular involvement should be recommended for all patients with MYH11 mutations. This is also clinically relevant as β-blockade may be considered in patients with TAAD and β-blockade may adversely affect systemic blood pressure and therefore perfusion of the brain.To date, a number of vascular disorders have been associated with mutations directly affecting SMC contractile proteins or proteins that disrupt SMC contractility (table e-1, ACTA2. The ACTA2 arteriopathy is characterized by an abnormally straight morphology of proximal branches of the circle of Willis, occlusive features, and a paucity of basal collaterals, distinct from classical “moyamoya.”5 The initial arterial morphology in the MYH11 patient also shows this straight configuration of the arterial circulation although, in contrast to ACTA2 patients, the index cases go on to develop profuse basal “moyamoya” collaterals over time. While in ACTA2 patients the distinctive radiologic signature is readily apparent,5 it may be that other SMC-related arteriopathies have morphologic signatures that are yet to be recognized.The radiologic phenotype of the observed arteriopathy appears to be different from that associated with MYH11 that we identified is likely to affect the communication between the motor domain and the coiled-coil tail of the SM-MHC-11 protein. Similar changes in the SM-MHC-11 protein structure in TAAD lead to reduced myosin motor elasticity, aberrant interactions with actin filaments, SMC shortening, and contractile force generation followed by upregulation of tumor growth factor–β activity.7 A similar mechanism may be implicated in the pathogenesis of the cerebral arteriopathy we observed with various external triggers contributing to disease development. The lack of a vascular phenotype in the father to date is in line with previous reports that suggested variability of disease onset and progression in relation to TAAD associated with MYH11.7The missense alteration, R1535Q, in exon 33 of MYH11 mutations and the need for broader than previously suggested vascular surveillance to include the cerebrovascular circulation. The contribution of MYH11 mutations to isolated cerebral arteriopathy remains to be established. We would propose that MYH11 testing could be considered in children with moyamoya who have atypical features on cerebral angiography or poor response to pial synangiosis.We emphasize the systemic nature of the vasculopathy associated with