Morphological and compositional analyses were carried out by using scanning electron microscopy (SEM) and energy-dispersive X-ray measurements (EDX). The optical and vibrational properties of LiAl(IO3)4 have been studied by UV-Vis and IR spectroscopy. LiAl(IO3)4 is found to crystallize in the non-centrosymmetric, monoclinic P21 space group, contrary to what was reported previously. Theoretical simulations and Rietveld refinements of crystal structure support this finding, together with the relatively high Second Harmonic Generation (SGH) response that was observed. Electronic band structure calculations show that LiAl(IO3)4 crystal has an indirect band gap 3)4 have also been calculated as a function of energy, as well as its elastic constants and mechanical parameters. LiAl(IO3)4 is found to be a very compressible and ductile material. Our findings imply that LiAl(IO3)4 is a promising material for optoelectronic and non -linear optical applications.Here we report on the non-hydrothermal aqueous synthesis and characterization of nanocrystalline lithium aluminum iodate, LiAl(IO Inorganic nanoparticles usually exhibit interesting magnetic, electrical, and optical properties, including non-linear optical (NLO) features. Many methods for the synthesis of inorganic nanoparticles exist, encompassing physical, chemical, and mechanical processes. The choice of the synthesis method is dictated by the structure that has to be obtained which, in turn, depends on the foreseen applications ,2. In faNon-centrosymmetric inorganic nanoparticles exhibit non-linear optical features (NLO), with generation of several harmonics and possible up-conversion of signals ,15. NanoHydrothermal and solvothermal synthesis methods are typically the most promising approaches for the preparation of metal iodate nanomaterials. These methods can be combined with microwave heating to improve the quality and the reproducibility of the final products .3)4, which is a novel and interesting material for optoelectronics and nonlinear optics. LiAl(IO3)4 nanocrystals have been synthetized using a soft-chemistry method, which takes advantage of the direct precipitation reaction between lithium iodate (LiIO3) and aluminum nitrate nonahydrate (Al(NO3)3·9H2O) from highly concentrated water solutions, without hydrothermal conditions. To the best of our knowledge, this is one of the first examples of such a simple chemical route for the preparation of iodate nanoparticles. The morphology of the final product was inspected by electron microscopy, and the average size of particles was measured by X-ray diffraction, using Le Bail fir refinement and the Williamson-Hall method (W-H). UV-Vis and infrared spectra were also measured and discussed. Preliminary results dealing with a crude estimation of the SHG efficiency of LiAl(IO3)4 were also included after comparison of the emitted signals from two colloidal suspensions, the reference sample being LiNbO3 nanocrystals of similar size. The optical band gap of LiAl(IO3)4 nanoparticles was measured experimentally by absorbance spectroscopy. The second part of the manuscript is devoted to the theoretical calculation of the structural model and physical properties of this new compound using the Density-Functional Theory (DFT).In the first part of this work, we describe for the first time a simple and inexpensive chemical route for the preparation of pure nanocrystals of LiAl and aluminum nitrate nonahydrate (Al(NO3)3∙9H2O, 98%), were used without further purification. Powder X-ray diffraction patterns (XRD) of the final product were obtained at ambient conditions using a Panalytical EMPYREAN MALVERN  powder diffractometer  with a 0.01° step size and an acquisition time of 6 s/step in the 17–85° 2θ range. The homogeneity and chemical composition of the sample were investigated by Scanning Electron Microscopy (SEM) using a Tescan Vega  electron microscope system attached to a Peltier-cooled XFlashTM  silicon drift detector, model 410 M, for energy dispersive X-ray (EDX) analysis. Optical absorption spectra were recorded using a Jasco V-630 UV-Vis  spectrophotometer in the 190–1100 nm wavelength range. Infrared spectra (FTIR) were recorded on a Jasco FT/IR-4200  setup between 1000 and 400 cm−1 using KBr pellets. Dynamic light scattering (DLS) measurements  were used to assess the colloidal stability and aggregation state of the synthesized samples after re-dispersion in ethanol.Reagents purchased from Sigma–Aldrich , lithium iodate (LiIOk-point grids in the first Brillouin zone (BZ). The geometry optimization was performed using the Broyden–Fletcher–Golfard–Shannon (BFGS) algorithm. The ultra-soft pseudopotential was used within the following valence electronic configuration: Li: 1s2 2s1; O: 2s2 2p4; Al: 3p1 4s2; and I: 5s2 5p5. For band structure calculations, in addition to the PBE functional we have implemented the nonlocal hybrid functional HSE06 (Heyd–Scuseria–Ernzerhof) , [010], and [001], are shown in We also studied the linear optical properties of LiAl(IO3)4 is a birefringent material, one of the most desirable features for active NLO materials [3)4, is 3)2) and lithium zinc iodate (LiZn(IO3)3), both crystallizing in the P21 space group, have similar birefringence indices at 3)4 is colorless, its transparency in the visible domain makes it a potential candidate for quadratic NLO applications and radio communications, in the special case of frequency duplication [The value of the aterials ,59. Baseectively . Incidenlication .Cij of the elastic tensor C of LiAl(IO3)4, as listed in Cij exist for LiAl(IO3)4. In order to be a considered as mechanically stable, a crystal structure at ambient pressure must fulfill the Born criterion, one of whose equivalent formulations is that the eigenvalues of the elastic tensor must all be positive [C tensor are shown in Finally, we also computed the components positive . The eigB), shear (G), and Young’s (E) moduli in the Voigt, Reuss, and Hill approximations, and the corresponding Poisson ratio P, were calculated and are summarized in B of LiAl(IO3)4 was found to be 28.7–36.1 GPa, indicating that LiAl(IO3)4 is a fairly compressible material. The obtained bulk modulus is comparable to that of Co(IO3)2 (B = 29.8 GPa) and Zn(IO3)2 (B = 21.6 GPa), which are structurally related to LiAl(IO3)4 [B/G is 1.17–126, suggesting that LiAl(IO3)4 is a brittle material [The computed elastic coefficients are of the same order of magnitude as other NLO materials . ElasticAl(IO3)4 ,43. On tAl(IO3)4 , and B/Gmaterial .3)4 nanocrystals, having potentially high IR NLO performances as exogenous optical biomarkers. The method makes use of a soft-chemistry technique, resulting within a few hours in precipitated nanoparticles from almost saturated solutions of the reagents. No hydrothermal processing is needed. The synthesized samples have been characterized by X-ray diffraction, scanning electron microscopy, EDX, UV-Vis, FTIR, and Second Harmonic spectroscopy. According to XRD results and DFT calculations, the P21 monoclinic structure was assigned to LiAl(IO3)4, instead of to the P63 hexagonal space group as previously proposed. Additionally, we also determined an average nanocrystal size at about 23 nm from Le Bail refinements and Williamson-Hall plots. The chemical composition and the expected stoichiometry of the nanocrystals were confirmed by both XRD and EDX. The optical band gap of the nanoparticles was estimated to be 3.43(3) eV, using absorption spectroscopy.We have reported a novel experimental approach for a facile and cost-effective synthesis of likely nontoxic LiAl(IO3)4. Theoretical calculations of the band structure showed that LiAl(IO3)4 is an indirect semiconductor with a calculated band gap Egap = 3.68 eV, close to the experimental value. Additionally, the theoretical study of the optical properties suggested that LiAl(IO3)4 has a birefringence index D = 0.21, close to that of the zinc iodate Zn(IO3)2.DFT simulations supported our findings about the crystal structure of LiAl(IO


Cognitive function is tested through speech- or writing-based neuropsychological instruments. The application and validity of those tests is impeded for patients with diseases that affect speech and hand motor skills. We therefore developed a “motor-free” gaze-controlled version of the Trail Making Test (TMT), including a calibration task to assess gaze accuracy, for completion by means of an eye-tracking computer system (ETCS). This electronic TMT version (eTMT) was evaluated for two paradigmatic “motor-neurodegenerative” diseases, Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). We screened 146 subjects, of whom 44 were excluded, e.g., because of vision deficits. Patients were dichotomized into subgroups with less  or severe motor affection . All 66 patients and all 36 healthy controls (HC) completed the eTMT. Patients with sufficient hand motor control  and all HC additionally completed the original paper–pencil-based version of the TMT. Sufficient and comparable gaze fixation accuracy across all groups and the correlations of the eTMT results with the TMT results supported the reliability and validity of the eTMT. PD+ patients made significantly more errors than HC in the eTMT-B. We hereby proved the good applicability of a motor-free cognitive test. Error rates could be a particularly sensitive marker of executive dysfunction. The assessment of cognitive functions can be problematic in patients with neurological disorders due to disease-related motor impairment ,2. NeuroWe have developed a digital version of the Trail Making Test (TMT) which is applied by means of an eye-tracking computer system (ETCS). The TMT is a widely used paper–pencil-based tool for the assessment of visual processing speed and executive functioning . In contThe purpose of our present study was to evaluate the usability and sensitivity of this electronic eye-tracking-based “motor-free” TMT-version (eTMT), which can be adapted to patients with different extents of motor impairment by using a fixed or a mobile and adjustable ETCS. Therefore, we examined patients with the two paradigmatic “motor” neurodegenerative diseases PD and ALS in early to advanced disease stages, also taking oculomotor functioning into account.Patients and healthy controls (HC) were prospectively recruited from a specialist outpatient clinic at the University Hospital Carl Gustav Carus Dresden, Germany and from a patient network . Patients with PD had an established diagnosis according to MDS clinical diagnostic criteria . ALS diaALS patients in LIS (in the ALS+ group) accustomed themselves to ETCS use as a communication device in daily life for at least two weeks before assessment.For all gaze data collection and test procedures, a monocular Eyegaze Edge remote IR eye-tracking device  with a sampling rate of 60 Hz was used, attached to the bottom of the stimulus screen. The ETCS screen was positioned fronto-parallel at a distance of 60–70 cm from the subject´s face. Patients with sufficient body control executed the tests seated and with the head rested upon a chin rest. All other patients were tested with a mobile and adjustable ETCS and either lying in bed or seated in a wheelchair see .Before completing the eTMT, participants had to pass a 13-point calibration task, which secured the minimum required fixation accuracy for the gaze-controlled operation of the ETCS in all four quadrants and thus all visual directions. Failure or trouble in this calibration task led to the cessation of study procedures.Fixation or gaze accuracy in the calibration task was analyzed as the average angular degree of deviance (°) between the target and gaze fixation points see .® eye-tracking data analysis suite . Selection of an item required a minimal fixation duration of 100 ms  with an error time of 550 ms . The dwell delay and dwell time scores were chosen according to previously published recommendations [Electronic gaze-controlled versions of both subtests of the TMT  were built, according to the original test form with regard to the layout of the test templates. As in the original test, they included exercise tasks preceding the respective subtest, each consisting of the first eight items of eTMT-A and eTMT-B see . The eTMndations ,18. VisuPatients with sufficient motor skills and all HC completed both the TMT (paper–pencil version) and eTMT in random order. Patients with severe motor impairment only completed the eTMT. For the written TMT, the completion times for both subtests and the ratio of the completion times were evaluated.SPSS Software  was used for statistical analysis. Group comparisons for categorical data were conducted using a chi-squared test. The normal distribution of the eTMT data was tested by a Kolmogorov–Smirnov test and the homogeneity of variance was tested by Levene’s test. For the between-group comparison of the eTMT-variables, a Kruskal–Wallis test instead of ANOVA was used if one or both assumptions were violated. Adequate post hoc procedures were performed as indicated and adjusted for multiple pairwise comparisons. Correlations between TMT and eTMT were calculated with Spearman’s rho. Significance level was set at α = 5%.n = 102 participants  completed the study procedure (p < 0.001) and Hoehn and Yahr stages in PD patients (p < 0.001) allowed us to categorize patients into two groups with respect to disease severity. ALS patients were grouped into patients with higher motor impairment (ALS+) and lower motor impairment (ALS−), depending on whether their ALSFRS-R-score was below/equal to or higher than 20, respectively. PD patients were categorized into patients with higher motor impairment (PD+) and lower motor impairment (PD−), depending on whether they reached a Hoehn and Yahr score equal to/higher than 2.5 or below 2.5, respectively. While ALS was sporadic for all patients in the ALS+ group, three patients in the ALS− group had a family history of ALS. Genetic testing of those familial ALS cases confirmed one case of genetic ALS with C9ORF72, while for the other two genetic testing revealed no mutation in known ALS genes. We screened 146 participants (105 patients and 41 HC) for eligibility, rocedure . As presF  = 1.44, p = 0.23) and thus for gaze accuracy in the calibration task TMT-B: r = 0.641, p < 0.001), the ALS- group ((e)TMT-A: r = 0.551, p = 0.009; (e)TMT-B: r = 0.526, p = 0.025), and the PD− group ((e)TMT-A: r = 0.551, p = 0.018; (e)TMT-B: r = 0.574, p = 0.010). No significant correlations between the test versions were found for the PD+-group ((e)TMT-A: p = 0.141; (e)TMT-B: p = 0.120).In total, 90 of the 102 enrolled participants were able to complete the written TMT, including only 2 of the ALS+ patients. The ALS+ group was therefore excluded from the respective analysis. Spearman’s rho revealed significant moderate correlations between TMT-A and eTMT-A b as wellF  = 1.78, p = 0.16), TMT-B  = 2.21, p = 0.09), or for the TMT-B/A ratio  = 0.37, p = 0.78).As presented in p = 0.044). Post hoc pairwise comparisons showed that PD+ patients made significantly more errors than HC in the eTMT-B  = 4.66, p = 0.003, ANOVA) and eTMT-B (H (4) = 21.31; p < 0.001; Kruskal–Wallis) (p = 0.006), to PD− , p < 0.01), to PD+ ; p = 0.008), and to HC , p < 0.001). For eTMT-B, Dunn–Bonferroni post hoc pairwise comparisons revealed significantly fewer fixations per second in the ALS+ subgroup than in HC  and in PD− . We found significant group differences for errors per fixation in eTMT-A (H (4) = 9.61, p = 0.047) and eTMT-B (H (4) = 11.53, p = 0.021; both Kruskal–Wallis) (p = 0.048).Significant differences could be demonstrated for the number of fixations per second in eTMT-A  g. Post h–Wallis) h. HoweveThe present study demonstrates that the ETCS-based assessment of cognitive functioning in motor-restricted patients via eTMT is feasible in a standard clinical setting, even in patients lacking experience with ETCS. In contrast to previous studies, a mobile and adaptable ETCS that could be used without a chin rest allowed for the examination of patients with very severe motor limitations—which is the subpopulation of patients for whom eye-tracking-based solutions for cognitive testing are most relevant and needed ,19. MoreLooking at the classic TMT parameters, however, it seems surprising that ALS patients did not perform significantly worse than HC in the present study, which is also contradictory to previous eTMT results . HoweverMost unexpected, however, was that PD patients did not perform significantly poorer than HC regarding the primary TMT outcomes of completion times and their ratio, either in TMT or eTMT, which is in contrast to a number of previous findings showing progressive cognitive decline in PD ,21. HoweThe finding of increased error rates of PD+ patients in eTMT-B, however, indicates the deterioration of cognitive control or executive functioning, which might be related to altered diffusion in the frontal lobe activity, in line with the development of fronto-temporal dysfunction in a relevant proportion of patients with PD ,22. ThisSevere ocular dysfunction and dyskinesia/tremor are major limitations of the presented technology. Moreover, we screened a relevant number of particularly PD patients who were too motorically impaired to perform the eTMT sitting in front of the ECTS and still too mobile (too much head movement) to complete it using the mobile ETCS version without a chin rest. Head-mounted systems might overcome these restrictions and even increase the usability of the approach . The lacBeside its small sample size, a main limitation of the study is a training effect for the gaze-controlled completion of the eTMT from part A to part B, particularly for ETCS-inexperienced subjects. This effect is strongly indicated by the comparison of the B/A ratio between eTMT and TMT: HC and PD patients need more time to complete eTMT-A than the TMT-A, while they present shorter completion times for eTMT-B than for TMT-B, and consequently lower B/−A ratios for the eTMT than for the TMT. This leads to the assumption that eTMT completion per se requires less time than TMT completion (gaze movements are quicker than hand movements), but that subjects lose time in eTMT-A for understanding and getting used to the dwell-time-based interaction. An important measure to solve or reduce this problem would be a more intensive ETCS training prior to the completion of eTMT. This could include, e.g., a writing task  and more importantly a longer TMT-specific practice task. This would allow participants to understand and to get used to the principles and the interaction speed of the unfamiliar task. This eTMT practice might consist of, e.g., writing a particular word by connecting letters or connecting colored circles in a given order, according to the principles and design of eTMT . Even then, however, it would be an important measure to collect normative data for the gaze-controlled eTMT, which is underlined by the comparison of TMT and eTMT data in the present study.In summary, we provide first evidence for the usability of a new motor-independent and valid cognitive assessment instrument using ETCS for classical movement disorders. Conclusions from this pilot study’s results are limited by its small sample size and the training effect discussed above. Another limitation with regard to the written TMT is the lack of availability of fine motor or particularly hand/arm motor function scores for our cohort, which thus cannot be taken into account as an influencing factor on performance in this original test version. However, and importantly, the bimodal grouping and the comparison of patients based on that was not meant to evaluate the influence of hand function, but to investigate whether overall motor disease progression is associated with progressed cognitive impairment, and whether patients with overall very severe motor impairment can still be properly assessed using the gaze-controlled version of the TMT. Therefore, the main outcome for all patients was their performance in the eTMT. The analysis of the correlation between the written and eTMT was additionally used to evaluate the validity of the latter. Nevertheless, the in-depth analysis of the effect of dominant hand/arm function on written TMT test results would be interesting to follow up in a larger cohort. Further research is needed to verify the findings by examining cognitive functioning assessed by the eTMT—improved as discussed above—in a larger sample of early-stage to late-stage ALS and especially PD. Ideally, future studies would have a longitudinal design and assess additional neurocognitive functions, including specific oculomotor functions like reflexive pro-saccade latencies. What would also be of interest are correlations between performance in eTMT with anti-saccade-error-rates as another indicator of executive dysfunction, e.g., ref. ,26 which


Reproduction and Fertility preservation series is composed of articles from experts on this topic and these will highlight key developments in fertility preservation and also identify the challenges that still face this exciting and relatively new field.Fertility preservation is a rapidly advancing field with numerous broad applications ranging from retaining the prospect of fertility in a child with cancer to protecting an entire species from extinction. In recent years, huge strides have been made in understanding the biology of male and female reproduction in animals and humans and using this knowledge to develop strategies for fertility preservation across a range of clinical and ecological applications. This  Reproduction and Fertility preservation series is composed of articles from experts on this topic: these will highlight key developments in fertility preservation and also identify the challenges that still face this exciting and relatively new field. Several leading authors in the field will be contributing, however, as this series is being published by Reproduction and Fertility, it is online and open access, and there is scope for more contributions. So do get in touch if you have something to enhance this excellent series in fertility preservation.Fertility preservation is a rapidly advancing field with numerous broad applications ranging from retaining the prospect of fertility in a child with cancer to protecting an entire species from extinction. In recent years, huge strides have been made in understanding the biology of male and female reproduction in animals and humans and using this knowledge to develop strategies for fertility preservation across a range of clinical and ecological applications. This what are we trying to preserve? While preserving germ cells is key, the approach to fertility preservation will depend on which germ cells are present – that is does the tissue contain sperm or not? Given the fundamental differences between the prepubertal and adult testis, consideration of age, pubertal stage and maturity are paramount when thinking about the options to preserve male fertility.When it comes to offering fertility preservation in males at risk of infertility, a key question to be asked is  in vitro fertilisation or intracytoplasmic sperm injection. Freezing semen should be offered to all men due to receive any treatment with a risk of impaired fertility (e.g. chemotherapy or radiotherapy). But what about when spermatogenesis is impaired? In this series, Dr Sarah Martins Da Silva will discuss the options and practicalities in this challenging situation.For adult males who face gonadotoxic treatments, cryopreservation of sperm containing semen is a proven option that will preserve the potential to father biological children, albeit with a requirement for assisted reproductive technologies (ARTs), which in its simplest form is intrauterine insemination or, the more involved, in vitro spermatogenesis must be translated from existing animal studies. Dr Jan-Bernd Stukenborg will describe how temperature may play a key role in this regard.Adolescence represents a difficult period when it comes to fertility preservation in males. Physical and emotional maturity of the patient plays a key role in what can be offered and important ethical considerations apply, for example, does the patient have the capacity to consent for treatment? Semen cryopreservation may be an option if the individual is well established in puberty, while surgical intervention to extract sperm can also be considered. However, for those whose testes do not yet make sperm (e.g. children), fertility preservation must focus on preserving the sperm precursor cells, the spermatogonial stem cells (SSC), in the tissue by cryopreservation of testicular tissue. While animal studies involving re-implantation of cryopreserved testicular tissues provide great promise for the future with sperm growing in the transplants . Despite the reliability of cryopreservation in preserving fertility, new techniques are being developed and will be discussed by Professor Pierre Comizzoli.Dr James Barrett will discuss the options and challenges in this context.Successful development of multiple fertility preservation options for indications such as prior to gonadotoxic treatments has lead clinicians and researchers to explore options in other patient groups such as those with disorders (or differences) in sex development . The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1).


Studies over the past four decades have elucidated the role of Wnt/β-catenin mediated regulation in cell proliferation, differentiation and migration. These processes are fundamental to embryonic development, regeneration potential of tissues, as well as cancer initiation and progression. In this review, we focus on the epigenetic players which influence the Wnt/β-catenin pathway via modulation of its components and coordinated regulation of the Wnt target genes. The role played by crosstalk with other signaling pathways mediating tumorigenesis is also elaborated. The Hippo/YAP pathway is particularly emphasized due to its extensive crosstalk via the Wnt destruction complex. Further, we highlight the recent advances in developing potential therapeutic interventions targeting the epigenetic machinery based on the characterization of these regulatory networks for effective treatment of various cancers and also for regenerative therapies. Sporadiing RNAs . Even thing RNAs .This review is specifically aimed at providing an overview of all well characterized epigenetic mechanisms which modulate the expression and function of the canonical Wnt pathway components and therefore affect Wnt target genes in various cancer types. In addition to this, the review will also cover advances made in the therapeutic interventions targeting the Wnt signaling pathway, especially through the epigenetic players.Wnt signaling is broadly classified into canonical and non-canonical Wnt signaling based on β-catenin -dependent and independent responses, respectively . CanonicMultiple factors have been shown to regulate the precise stimulation and inhibition of Wnt signaling. For instance, the R-spondin/LGR/RNF43 module is the key signaling paradigm that potentiates Wnt signaling. R-spondin family proteins regulate Wnt signaling via a common mechanism involving interaction with the Leucine-rich repeat-containing G-protein coupled (LGR) family of receptors . In the + signaling pathways, has been shown to converge with multiple pathways and regulate a diverse number of cellular processes required for development and organogenesis. The combination of receptor/co-receptor at the plasma membrane binding to Wnts acts as a regulatory switch to activate either β-catenin-dependent or β-catenin-independent pathways. In the PCP pathway, Wnt ligands, Wnt5a and Wnt11 bind to receptors FZD3 or FZD6 along with tyrosine kinase co-receptors ROR1, ROR2 or RYK to orient toward Wnt/PCP signaling , 1,2 diacylglycerol (DAG), and Ca2+ from the membrane-bound phospholipid phosphatidylinositol 4,5-bisphosphate via the action of membrane-bound enzyme phospholipase C , thereby regulating cell adhesion and cell migration  and Caignaling . Subsequignaling . This caignaling . The Wntignaling . In the lipase C . The netigration .APC gene are mutated, especially in the region required for interaction with the armadillo (arm) repeats of β-catenin  inhibited the nuclear accumulation of β-catenin  knockout mice  group of proteins are involved in embryogenesis. In adults, these are required for maintenance and homeostasis. HOXA5 has been shown to coordinate with the Wnt pathway in driving stem cell differentiation. HOXA5 acts as a tumor suppressor and promotes intestinal cell differentiation by inhibiting the Wnt/β-catenin pathway. Wnt pathway, on the other hand, inhibits HOXA5 via MYC which is a direct target of the Wnt signaling . Anotheregulated .+ intestinal stem cells (The Specificity Protein (SP) family of zinc finger proteins have been shown to regulate Wnt signaling as well. SP1, one of the earliest transcription factors to be identified, has been shown to regulate the stability of β-catenin by modulating its interaction with the Wnt destruction complex. Interestingly, SP1 depletion led to an increased phosphorylation and ubiquitination of β-catenin in colorectal cancer cell lines . SP5, onem cells . Anotherem cells .de novo methylation while DNMT1 is required for maintenance and is critical for the inheritance of DNA methylation  is enriched at the active promoters whereas trimethylation of histone H3 lysine 27 (H3K27me3) and histone H3 lysine 9 (H3K9me3) are typically seen at inactive promoters. The enhancers are enriched with histone H3 lysine 4 monomethylated (H3K4me) and acetylated histone H3 lysine 27 (H3K27Ac) modifications. The histone methyltransferases (KMTs) and histone demethylases (KDMs) add and remove methyl groups to/from histones respectively. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) add and remove acetyl groups to/from histones, respectively . The incMicroRNAs (miRs) are small non-coding RNAs typically consisting of 20–21 nucleotides and are essential for various developmental processes and diseases. They regulate gene expression post-transcriptionally either by inhibiting translation or inducing mRNA degradation . AnotherWnt signaling is a complex pathway modulated by various cellular and environmental cues to precisely regulate the myriad of cellular processes during development and disease. Wnt driven cancers have either hyperactivation of positive regulators or hypoactivation of negative regulators. In this section, we provide the details on the epigenetic modulations undergone by the components of the Wnt pathway. WNT5A, WNT9A, and WNT10B, suggest a potential tumor suppressive role , an adaptor molecule known to suppress Wnt signaling has been observed to be downregulated in colon cancer cells and is associated with decreased survival and increased intestinal tumor burden  asymmetric methylation. The advantage of such a poised nature of chromatin is to enable synchronous and rapid activation of tissue specific genes  . AberranTargeting the Wnt pathway is an attractive model for therapeutics and cancer therapy. Aberrant activation of Wnt signaling is associated with poor cancer outcomes and recent strategies involve targeting the pathway in diverse cancers . InhibitThe downstream signaling network of Wnt signaling pathway involves β-catenin nuclear signaling through interaction with various nuclear factors to drive target gene expression and tumorigenic phenotypic changes. Several attempts have been made to target this β-catenin and TCF interface to modulate target gene expression and induce regression of tumors. For instance, PKF115-584, CGPO49090, and PKF222-815 are fungal derived drugs and inhibit β-catenin’s interaction with TCF. These are currently in phase I and phase II of clinical trials . High thThe interaction of β-catenin with various co-factors is crucial for potentiating the Wnt pathway driven gene expression and associated cellular consequences, especially when the pathway is hyperactivated, has also been exploited to design drugs for cancer treatment. For instance, PRI-724 disrupts the interaction between β-catenin and its co-transcriptional activator CBP . SeveralIn this review, we have provided an overview of the regulation of the Wnt signaling pathway in various cancer types, with a special emphasis on the crosstalk with the epigenetic machinery. Apart from undergoing alterations at the genetic level, the Wnt signaling pathway components are dysregulated due to various ncRNAs and chromatin modifiers. In turn, the Wnt effector β-catenin is also involved in various epigenetic processes due to its interaction with a multitude of epigenetic players. We have also provided an overview of the signaling pathways that work in conjunction or restrict the activity of the Wnt signaling pathway.Aberrant Wnt pathway activation seems to be the root cause for the development and progression of multiple cancers. However, even four decades after the discovery of the Wnt signaling, therapeutic intervention of this key pathway remains far from achieving its potential. Several factors seem to be responsible such as: the pathway is intricate and regulated by multiple combinations of Wnt ligands and their receptors, crosstalk of canonical and non-canonical pathways, and crosstalk with other signaling pathways. Further, multiple mutations of the Wnt signaling components cause constitutive activation of β-catenin, making it difficult to target the upstream players. Additionally, the physiological roles of the Wnt/β-catenin signaling are important for normal cellular functions. It is the combination of various signaling pathways that results in an imbalance in the rate of activation. Hence, future therapeutics will be aimed at optimizing the rate of activation and directing it toward maintaining the cellular homeostasis.in vivo  have emerged as an efficient technology to degrade proteins of interest in vivo . One sucant mice . Similarant mice . RecentlWnt signaling crosstalks with several signaling and regulatory networks including epigenetic networks, mechano-sensing and environmental cues to modulate the expression of target genes crucial for developmental paradigms and tumorigenesis. Hence, holistic understanding of the molecular changes acquired during the initiation of Wnt signaling driven cancers is critical for disease etiology and future therapeutics. Identifying the pioneering molecular cues will be useful for developing better cancer therapies. The role of nuclear factors driving the Wnt/β-catenin signaling response raises hope of using drugs targeting these effectors. Obtaining a better picture of protein-protein interaction complexes regulating Wnt/β-catenin signaling will be helpful in identifying future therapeutic targets. Understanding the comprehensive picture of nuclear regulators and/or missing partner(s) crucial for convergence with other pathways will be essential for the treatment of Wnt signaling related diseases.All the authors contributed to design the concept and structure of the review article, and approved the submitted version. AS and RM collected and analyzed the literature, and wrote bulk of the manuscript. AS prepared the figures. RM and SG provided critical inputs as the corresponding authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Uric acid (UA), a known danger signal released from injured cells, is a valuable sign of inflammation. We aimed to evaluate the association of serum UA levels before the start of conditioning regimens with the risk of hepatic sinusoidal obstruction syndrome (SOS) development after hematopoietic stem cell transplantation (HSCT).Two hundred and twenty-two children who underwent allogeneic HSCT at the Pediatric BMT Unit of Hacettepe University between 2000 and 2014 were included in this retrospective study. Serum UA levels were measured before conditioning as an indicator of the pre-transplant inflammatory status of the patients. Patients with and without a diagnosis of SOS were compared regarding primary diagnosis, previously described risk factors for SOS, and pre-conditioning serum UA.SOS was diagnosed in 42 patients who had higher pre-conditioning serum UA levels compared to those who did not. Pre-transplant serum creatinine, gamma-glutamyl transferase, bilirubin, ferritin, and C-reactive protein levels did not differ significantly among patients with and without SOS; however, serum albumin was lower in the patients who developed SOS. Receiver operating characteristic analysis revealed that a pre-conditioning UA level higher than 3.32 mg/dL was predictive of SOS. When applied to a multivariate model, only pre-conditioning UA and albumin levels remained significant risk factors for SOS .Our results suggest that pre-conditioning serum UA is an independent risk factor for SOS, and it might be used as an early predictor of hepatic SOS together with previously described clinical and laboratory parameters. Hepatic sinusoidal obstruction syndrome (SOS), also known as hepatic veno-occlusive disease, is a serious complication of hematopoietic stem cell transplantation (HSCT) and is clinically characterized by fluid retention, painful hepatomegaly, and hyperbilirubinemia. It is the most frequent and well-studied of the early-onset vascular endothelial syndromes that develop after HSCT. It occurs in 5%-15% of patients after allogeneic HSCT. Variations in its incidence are attributed to multiple factors such as the diagnostic criteria used, center experience, year of HSCT, and patient type . PatientSOS is initiated with the sinusoidal endothelial cell damage caused mainly by cytotoxic effects of intensive conditioning regimens including chemotherapy and/or radiotherapy given before transplantation. Prophylactic immunosuppressive therapies, growth factors used to support engraftment, developing infections, and transplantation itself can also cause endothelial damage ,5. CliniTwo hundred and twenty-two children  who underwent allogeneic HSCT in the Pediatric BMT Unit of Hacettepe University between 2000 and 2014 were included in the retrospective data analysis. Serum UA levels measured before the initiation of the conditioning regimen (day -9) and after the conditioning regimen was completed (day 0 before transplantation) were analyzed to assess any changes in serum UA that would be indicative of the pre-transplant inflammatory status of individual patients. Pre-conditioning serum creatinine, transaminases, gamma-glutamyl transferase (GGT), and total bilirubin levels were recorded for all patients together with iron overload and cytomegalovirus (CMV) serology for the exclusion of other potential causes of hyperuricemia and hepatic dysfunction. Inflammatory markers  were also noted at the same time point. The association of serum UA levels of the pre-transplant period with the development of SOS was assessed. HSCT was performed according to standard institutional transplantation procedures. The patients received either myeloablative (intravenous busulfan-based with no area under the curve [AUC] targeting) or reduced-intensity conditioning (fludarabine-based) regimens depending on the primary disease or disease status. GVHD prophylaxis consisted of cyclosporine and methotrexate with or without rabbit anti-thymocyte globulin. Enoxaparin, vitamin E, and ursodeoxycholic acid were administered for SOS prophylaxis to all patients beginning with conditioning. None of the patients received allopurinol before or after transplant. Defibrotide was started when the clinical manifestations of SOS developed with the exclusion of other potential diagnoses. Hepatic SOS was diagnosed when two or more European Society for Blood and Marrow Transplantation (EBMT) diagnostic criteria  were present and the EBMT severity grading system was used ,13. The The associations between clinical variables including patient- and transplant-related risk factors  and hepatic SOS were analyzed using a logistic regression model. Only the variables with p<0.2 in the univariate analysis were subjected to multivariate analysis. The variables with p<0.05 were considered significant. Comparisons within and between the groups were performed using the Wilcoxon signed-rank and Mann-Whitney U test, respectively. Median follow-up time and overall survival were estimated using Kaplan-Meier limit estimation. The Cox proportional hazards model for multivariate analyses of survival was used. Receiver operating characteristic (ROC) curve analysis was performed to calculate sensitivity, specificity, and AUC for peak pre-conditioning serum UA of ≥3.32 mg/dL.The patient and transplant characteristics are summarized in When the median serum UA levels of all patients were evaluated, pre-conditioning UA levels were found to be significantly higher than the post-conditioning levels . After grouping patients by the development of hepatic SOS, the 42 patients (19%) who developed mostly mild/moderate hepatic SOS (91%) were found to have higher median pre-conditioning UA levels compared to those who did not develop SOS . Pre-transplant serum creatinine, GGT, total bilirubin, ferritin, CRP, and albumin were checked, and while most of these variables did not differ significantly regarding SOS groups (p>0.05), albumin was found to be lower in patients with SOS compared to those without SOS (p=0.02) . Among tWhile a significant decrease in the serum UA levels of the patients who did not develop SOS was observed on the day of HSCT following conditioning , there was no significant change in the serum UA levels of patients who developed SOS . The development of SOS was associated with higher pre-conditioning UA levels (p=0.002). The ROC curve was drawn to determine a cutoff value and evaluate the predictive value of pre-conditioning UA for hepatic SOS development. As shown in =0.35).The probability of 10-year overall survival after HSCT was 75% for all patients enrolled with a median survival of 114 months. There was a significant difference between survival rates of patients who developed SOS  and those who did not  (p=0.047). However, the cutoff value of pre-conditioning UA of 3.32 mg/dL had no significant effect on the survival of the patients -1, and IL-6 have been reported to activate xanthine oxidase, thus stimulating UA production, which is involved in the pathogenesis of early non-infectious transplant complications, such as SOS. Even in the absence of clinically defined hyperuricemia (serum UA of >7 mg/dL) based on the solubility limit of urate in body fluids, this positive feedback loop may cause a further release of cytokines and endogenous adjuvants that contribute to the development of endothelial cell injury in an inflammatory setting ,25, as iOne of the limitations of our study is its retrospective nature. Therefore, serial measurements of serum UA and other pro-inflammatory and/or endothelial injury markers over the transplant course could not be performed. Also, the design of our study did not allow us to investigate whether UA plays an active role in the pathogenesis of hepatic SOS through induction of a non-infectious inflammatory reaction in the allogeneic setting or is simply a biomarker of inflammatory status. Since our patient population mostly included pediatric patients who received bone marrow grafts from matched related donors after myeloablative conditioning for non-malignant disorders, we could not draw any conclusion about the impact of serum UA on SOS development in different transplant settings such as unrelated and haploidentical transplants, which carry a higher risk for SOS. However, we think that the association of serum UA higher than the cutoff value with SOS in such a restricted population could be accepted as proof of its predictive strength.This is the first report about the association of pre-conditioning serum UA level and hepatic SOS in HSCT recipients and it supports the use of pre-transplant serum UA level as a risk factor for SOS. There is a need for mechanistical studies to understand the precise role of UA in the pathogenesis of SOS as an inflammatory mediator in the allogeneic transplant setting. Further studies with independent cohorts and in different transplant settings may also help to clarify the role of UA in predicting high-risk patients for SOS together with other defined clinical and laboratory markers of endothelial injury.


Research Design and Methods. A retrospective case-control study with a total of 945 children, including 471 patients with T1D and 474 healthy siblings, was performed. Participants were volunteers from the Germans Trias i Pujol Hospital and DiabetesCero Foundation. Parents of children enrolled in the study completed a questionnaire that included questions about weeks of gestation, preterm delivery risk, weight at birth, and prenatal betamethasone exposure of their children. Multiple logistic regression was used to detect the association between betamethasone exposure and T1D.  Betamethasone, a glucocorticoid used to induce lung maturation when there is a risk of preterm delivery, can affect the immune system maturation and type 1 diabetes (T1D) incidence in the progeny. It has been described that prenatal betamethasone protects offspring from experimental T1D development. The main aim of this study was to evaluate the possible association between betamethasone prenatal exposure and T1D in humans. p = 0.139). The percentage of betamethasone-treated subjects with T1D in the preterm group  was significantly higher than that found in the control group  (p = 0.003). The odds ratio for T1D associated with betamethasone in the univariate logistic regression was 0.59  and in the multivariate logistic regression was 0.83 .  We compared T1D prevalence between subjects prenatally exposed or unexposed to betamethasone. The percent of children with T1D in the exposed group was 37.5% (21 of 56), and in the unexposed group was 49.52% (410 of 828) ( The results demonstrate that the prenatal exposure to betamethasone does not increase T1D susceptibility, and may even be associated with a trend towards decreased risk of developing the disease. These preliminary findings require further prospective studies with clinical data to confirm betamethasone exposure effect on T1D risk. Although nonsignificant, a trend towards a protective effect of betamethasone against T1D development was observed . By contrast, the effect of weeks of gestation on T1D showed a statistically significant OR of 1.11   for T1D associated with betamethasone was 0.59  .In this study, we address for the first time the relationship between prenatal administration of betamethasone and T1D development in the offspring. Previous results of our group demonstrated a protective role of prenatal betamethasone exposure against T1D in NOD mice . The chon = 945), Greene et al. started with approximately 500,000 children born in Denmark in a specific period (between 1 January 1997 and 31 December 2004). So, our cohort is smaller than the Danish one but includes 50% of T1D patients, in comparison to less than 1% of Danish children with T1D. Importantly, in the Danish cohort the authors took into account not only prenatal betamethasone administration but also glucocorticoid exposure  throughout the pregnancy. Only 4.5% of the prenatally glucocorticoid-exposed children received one or more systemic glucocorticoids (betamethasone and other synthetic glucocorticoids). In this sense, we believe that our study was focused on the effect of a single course of prenatal systemic betamethasone exposure in the third trimester. The duration of the exposure before giving birth could prove crucial because betamethasone and its derivatives have a half-life of approximately 3 days in the human body [β-cell growth, metabolism, and immunogenicity [β-cells. These effects have positive consequences on self-tolerance in NOD mice, in this regard, betamethasone might even be a factor that could help to tip the balance towards β-cell tolerance, thus contributing to prevent autoimmunity in subjects at risk of developing T1D.The effects of glucocorticoids, administered during pregnancy, on childhood diabetes were previously reported in a Danish cohort study . This stman body . We hypoman body , 32. On man body  and modiman body . Our recgenicity  which coInterestingly, and independently of the effect of betamethasone, the T1D group of this study shows a longer pregnancy duration than the control group. Other studies do not report any effect  or pointWe are well aware of the limitations of our study, including the use of a questionnaire instead of clinical data registry. However, due to technical and ethical issues, it was impossible to link both the medical registries of the mothers and their children in our study population. Another limitation was the recall bias in the questionnaire, particularly for the betamethasone exposure, which is our main focus. We have determined that 5% of mothers do not remember whether they received betamethasone. To avoid it, we have defined a specific target population, and the questionnaire has been designed as short, precise, and accessible as possible. Nonetheless, this “missing” group gives similar perinatal data than the unexposed betamethasone group, so it is unlikely that group participants received betamethasone at late pregnancy. In addition, participants were recruited from two sources, but the incidence of T1D in the two geographical areas where the questionnaire was performed is similar  , 38. TheHowever, additional results from the present study fit well with previous reports, thus validating the study group. First, a significantly higher number of preterm newborn males was observed as compared to their female counterparts. This finding has already been reported in previous studies , 40. SecOur study suggest that the prenatal exposure to betamethasone does not increase the susceptibility to T1D, and may even be associated with a trend towards decreased risk of developing this disease. However, these preliminary findings require further prospective studies with clinical data registries involving a larger sample size to draw definitive conclusions regarding the effects of prenatal betamethasone exposure on the risk of developing T1D in later life.


To summarize the existing knowledge on fall risk associated with antidepressant use in older adults, including the underlying pathophysiology, and assist clinicians in (de-) prescribing antidepressants.Untreated depression and antidepressant use both increase fall risk in older people. Antidepressants differ in fall-related side effect profile. They contribute to (or cause) falling through orthostatic hypotension, sedation/impaired attention, hyponatremia, movement disorders and cardiac toxicity. Although withdrawal of antidepressants is recommended in fall-prone elderly persons, physicians are frequently reluctant to deprescribe antidepressants. Practical resources and algorithms are available that guide and assist clinicians in deprescribing antidepressants.Insight in fall-related side effect profile of antidepressants, and clinical decision tools such as the STOPPFalls antidepressant withdrawal algorithm assist prescribers in rational (de-) prescribing decision making. The aim of this clinical review was to summarize the existing knowledge on fall risk associated with antidepressant use in older adults, describe underlying mechanisms, and assist clinicians in decision-making with regard to (de-) prescribing antidepressants in older persons.We comprehensively examined the literature based on a literature search in Pubmed and Google Scholar, and identified additional relevant articles from reference lists, with an emphasis on the most commonly prescribed drugs in depression in geriatric patients. We discuss use of antidepressants, potential fall-related side effects, and deprescribing of antidepressants in older persons.Untreated depression and antidepressant use both contribute to fall risk. Antidepressants are equally effective, but differ in fall-related side effect profile. They contribute to (or cause) falling through orthostatic hypotension, sedation/impaired attention, hyponatremia, movement disorder and cardiac toxicity. Falling is an important driver of morbidity and mortality and, therefore, requires prevention. If clinical condition allows, withdrawal of antidepressants is recommended in fall-prone elderly persons. An important barrier is reluctance of prescribers to deprescribe antidepressants resulting from fear of withdrawal symptoms or disease relapse/recurrence, and the level of complexity of deprescribing antidepressants in older persons with multiple comorbidities and medications. Practical resources and algorithms are available that guide and assist clinicians in deprescribing antidepressants.(De-) prescribing antidepressants in fall-prone older adults is often challenging, but detailed insight in fall-related side effect profile of the different antidepressants and a recently developed expert-based decision aid STOPPFalls assists prescribers in clinical decision-making. In Western societies, over one-third of community dwelling and over 40% of institutionalized older persons fall every year . The con. Psychotropic medication, including antidepressants, has consistently been associated with falls. A recent systematic review and meta-analysis published by our group showed an odds ratio (OR) of 1.57 for antidepressant-related falls [In general, falls are multifactorial. Over 400 fall risk factors have been identified, of which the most important modifiable risk factors are mobility problems and the use of fall-risk increasing drugs (FRIDs) . With thDepression is a common and important cause of morbidity and mortality in older people worldwide, affecting around 10–15% of community-dwelling older persons . In the Antidepressant use is also common in older persons; almost 10% of community-dwelling older people , and oneAlthough often overlooked, fall incidents are among the most important adverse drug effects of antidepressants in the older population. Given the above-mentioned potential consequences of falls in older persons on morbidity and mortality, fall prevention is essential in this population. Medication review is a crucial element of the multifactorial fall assessment, especially if the patient uses psychotropic medicines. If clinical condition allows, withdrawal of psychotropic drugs is recommended . The proAs stated above, prevalence of both depression, accompanying antidepressant use and falls are common in older people. In an effort to prevent future falls and protect function and quality of life, judicious prescription of antidepressants is essential. In clinical practice, however, these (de-) prescribing decisions are often highly complex, highlighting the need for detailed knowledge of the risks and benefits of prescribing and deprescribing of this drug class. In this clinical review, we give an overview of the current literature regarding antidepressant use and antidepressant withdrawal in the light of fall risk. This clinical review was informed by a literature search conducted in May 2020 in Pubmed and Google Scholar with citation and reference checking. Personal reference libraries and antidepressant SmPCs (summary of product characteristics) were also utilized. Keywords for the searches included “falls”, “antidepressant”, “deprescribing” and “older adults” (and appropriate variations). We do not provide an exhaustive systematic review of the literature on all antidepressants and their relationship to falling, but merely focus on the most commonly prescribed drugs in geriatric depression. As such, antidepressants in general are discussed while examples of medication classes are provided to illustrate the potential fall-related safety concerns. We intend to call attention to the increased fall risk associated with both depression and antidepressant use in the aged. Also, we intend to assist clinicians in appropriate use of antidepressants in older persons with increased risk of falling.The first step in performing a medication review aimed at reducing fall risk is to thoroughly review all current medications for their (contra-) indications, including the antidepressant history , use of over-the-counter drugs and as required medications, supplements, alcohol use and medications not taken orally or in solid form . Fall ri. In fact, cognitive and behavioral therapy for insomnia (CBT-I) is generally effective in primary, and secondary insomnia (in patients with comorbid depression): it improves depression and anxiety symptoms [Pharmacological treatment of depression is indicated in case of a major depressive disorder. In mild to moderate severity, psychotherapy is recommended as an initial treatment option . Althougsymptoms . Therefowww.switchwiki.eu are used by professionals from over 130 countries worldwide.For most antidepressants, lowest effective and maximal dose for geriatric patients have been established (SmPCs and handbooks). Dose adjustment can be indicated in case of decreased glomerular filtration rate in patients using drugs that have a small therapeutic index and are cleared renally (e.g. lithium) . SSRI asIn current clinical practice, despite existing guidelines advising a stepwise order, the next line(s) of antidepressant therapy are frequently selected by means of educated trial-and-error, and/or titrated through therapeutic drug management (TDM). This approach is associated with disappointing success rates , delays n = 181, mean age 66 years old) showed that adding an atypical antipsychotic (aripiprazole) to an antidepressant (venlafaxine) resulted in remission in 44% of the participants compared to 29% percent in the (add-on) placebo group  [If switching to another agent also fails to achieve remission, add-on therapy should be considered. Lithium augmentation is effective in older adults, with an overall response rate of 42%  . Of noteOur recent systematic review and meta-analysis , includiUnfortunately, most of the evidence on antidepressant-related fall risk in older adults originates from observational studies comparing antidepressant users versus non-users; published data on antidepressant head-to-head comparison studies in older adults are scarce. Therefore, the evidence in this field may be biased through confounding by indication. It is, however, well established that adverse effect profile and tolerability differs substantially between different (groups of) antidepressants Table . In clinSedation  is a frequent side effect of antidepressants and increases fall risk by impaired reaction capability . Many anSSRIs (and related drugs) have alerting properties and may cause nocturia, impairing sleep and subsequently daytime drowsiness . TheoretOrthostatic hypotension  is highAnticholinergic medication exposure increases fall risk directly through negative effects on cognitive and physical functioning, because it may cause postural hypotension, impaired cognitive performance, arrhythmia, QT prolongation, and blurred vision . AntichoTCAs, SSRIs, and lithium have dopamine blocking effects, and can, therefore, induce drug-induced Parkinsonism, characterized by tremor, bradykinesia, hypokinesia, rigidity, and postural instability. Drug-induced Parkinsonism can occur both shortly after antidepressant initiation (within days), and in chronic use (after months), albeit to a lesser extent. The prevalence of these movement disorders associated with SSRI use in older persons is approximately 10% . ExtrapyAntidepressant use  is freqAlthough newer generation antidepressants were initially reported to have fewer and less severe cardiovascular adverse effects, current evidence shows that clinically relevant cardiovascular (event) risk is equal for TCAs, SSRIs and other antidepressants , 50. SSRBupropion and mirtazapine also appear to have little cardiovascular side effects . Also, tIn older people, hyponatremia is a frequently occurring and potentially serious adverse drug effect of antidepressants . HyponatLiterature shows that SSRI and TCA use may negatively impact bone mineral density, and increase (hip) fracture risk . This inHypericum Perforatum) or lithium, there is a risk of developing the potentially life-threatening serotonin syndrome, albeit rare [In general, antidepressants are characterized by having a high risk of drug–drug interactions, obviously limiting treatment options in older individuals with comorbidity and polypharmacy. The tendency to cause drug–drug interactions however, varies considerably between antidepressants. In general, older compounds, such as TCAs and MAOIs, have a higher potential for these interactions than newer compounds . Of the eit rare . This syIn general, older adults with polypharmacy are open to medication deprescribing if their doctor thinks it appropriate . AchieviLiterature on how to taper antidepressants is scarce and international guidelines generally do not provide detailed guidance with regard to dosing steps and rate of tapering. Instead, they recommend “slow” tapering. In current clinical practice, dose tapering in weekly steps is often performed for SSRI/SNRIs, based on their elimination half-life. All SSRIs and SNRIs (except from fluoxetine and vortioxetine) have elimination half-lives not exceeding 40 h, and, therefore, steady-state concentration is reached within 1 week after dose adjustment. Slow tapering, however, may be challenging because antidepressants come in only a limited number of registered dosages that do not allow for flexible and individualized slow and gradual tapering over time . EspeciaAnother aspect to consider is the timing of deprescribing. Stress-inducing circumstances and insomnia reduce likelihood of successful antidepressant withdrawal . Thus, iAlso among prescribers there is reluctance to deprescribe antidepressants, despite the fact that its use is a well-established and common fall-risk factor, and deprescribing of FRIDs and specifically antidepressants is an effective means of reducing fall risk . This rehttps://www.eugms.org/research-cooperation/special-interest-groups/falls-and-fractures.html). The STOPPFalls tool is officially endorsed by the EuGMS and by the STOPP-START group. Whether implementation of this tool in clinical practice translates to reduction in fall risk is an area of ongoing research.A number of tools are available to assist clinicians in deprescribing potentially inappropriate medications in older people, including FRIDs . These tDepression and antidepressant uses are common in older people. Both untreated depression and antidepressant use contribute to fall risk. Depression increases fall risk through psychomotor retardation, deconditioning, gait/balance abnormalities, impaired sleep/attention and fear of falling. Antidepressants are FRIDs and contribute to (or cause) falling through causing sedation, impaired balance/reaction time, OH, hyponatremia, cardiac conduction delay/arrhythmia, and/or drug-induced Parkinsonism.Falling is an important driver of morbidity and mortality that requires prevention in older people. To minimize fall risk, it is important to make a personalized assessment, prescribe antidepressants with caution, and deprescribe antidepressants when the  risks of antidepressant use outweighs the  benefits.A major barrier for deprescribing antidepressants is the high level of complexity of deprescribing antidepressants in older persons with multiple comorbidities and medications. However, detailed insight in fall-related side effect profile of the different antidepressants and the newly developed expert-based decision aid assist in clinical decision-making.


Mixed economies provide a unique context for testing theories of fertility change. Because they have a stake in two traditions, mixed-economy households balance the demands of both a labor-based subsistence economy, which benefits from a large family, and a wage-labor economy, which benefits from reduced fertility. Additionally, household size changes over the course of its life-cycle and shapes available economic opportunities. Here we argue that in mixed economies, fertility may reflect opportunities for livelihood diversity rather than simply responding to the restricted socioeconomic benefits of small families. While low fertility may in some cases have an economic benefit, low fertility can also limit the livelihood diversity of a household which is a key strategy for long-term economic success. We test this prediction with longitudinal data from a Maya community undergoing both a sustained decline in fertility and rapid integration into the market economy. Using household-level fertility, number of adults, and livelihood diversity at two time points, we find that household size is positively related to livelihood diversity, which in turn is positively related to household income per-capita. However, household size also has a negative association with income per capita. The results reflect a balancing act whereby households attempt to maximize the economic diversity with as few members as possible. Broadly, these results suggest that theories of fertility decline must account for how households pool resources and diversify economic activities in the face of increasing market integration, treating fertility as both an outcome and an input into economic and reproductive decision-making. How households transform over the course of economic and social transitions has long been a central question of social sciences. Under its various monikers of modernization, industrialization, urbanization, and market integration, studies of the cascading effects of market involvement on indigenous and small-scale communities have had consequences on resource use , health In the case of fertility, motivated by an evolutionary puzzle , 12 whermixed economies, where households combine subsistence production and wage-labor. In mixed economies, households not only engage in both the market economy and subsistence production, they also maintain both traditional sharing and cooperative relationships , and livelihood diversity is positively related to number of adults . By contrast, for new households, livelihood diversity  and number of adults  showed no significant association with income per capita, though the size and sign of the associations are suggestive. Finally, for established households, household age was positively associated with fertility in 2010  while age showed no association with fertility in new households . Furthermore, when household age was restricted to just women, age showed positive association with completed fertility for established households  but showed no association within new households .The results of the full SEM show the contrasting effects of livelihood diversity and number of adults on income per capita , Fig 2. The stratified SEM shows how fertility and livelihood diversity shapes income per capita over the course of the household life cycle . We chosFor established households, results are qualitatively similar to the full mode. Livelihood diversity is positively associated with income per capita , while number of adults is negatively associated with income per capita , however the coefficient falls just outside of statistical significance. As in the full model, number of adults is positively associated with livelihood diversity . Additionally, the effects of fertility in 2010 shows a positive association with number of adults in 2017 .For new households, results are also largely consistent with the full model. First, livelihood diversity shows a positive association with income per capita  and number of adults shows a negative association with income per capita . These results contrast with those of the bivariate analyses, where these relationships were of similar sign and magnitude, yet failed to reach statistical significance. In the full model, accounting for all variables simultaneously provides a better assessment of the combined effect of number of adults and livelihood diversity on income per capita. Additionally, number of adults maintains a positive association with livelihood diversity  indicating that the more adults living in newly established households, the more economic activities these households are engaged in. For new households, however, the total fertility of women in 2010 was not a significant predictor of the size of the newly formed household in 2017 . Sensitivity analyses using completed fertility and fertility of just the female head of household are qualitatively similar to the main models presented here Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Microglia are integral mediators of innate immunity within the mammalian central nervous system. Typical microglial responses are transient, intending to restore homeostasis by orchestrating the removal of pathogens and debris and the regeneration of damaged neurons. However, prolonged and persistent microglial activation can drive chronic neuroinflammation and is associated with neurodegenerative disease. Recent evidence has revealed that abnormalities in microglial signaling pathways involving phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) may contribute to altered microglial activity and exacerbated neuroimmune responses. In this scoping review, the known and suspected roles of PI3K-AKT signaling in microglia, both during health and pathological states, will be examined, and the key microglial receptors that induce PI3K-AKT signaling in microglia will be described. Since aberrant signaling is correlated with neurodegenerative disease onset, the relationship between maladapted PI3K-AKT signaling and the development of neurodegenerative disease will also be explored. Finally, studies in which microglial PI3K-AKT signaling has been modulated will be highlighted, as this may prove to be a promising therapeutic approach for the future treatment of a range of neuroinflammatory conditions. Defined as an inflammatory response within the central nervous system (CNS), “neuroinflammation” refers to activation of the brain’s innate immune system following a disturbance to homeostasis, such as injury, infection, or the onset of neurodegeneration, which frequently occurs with aging. This inflammatory response represents a highly sophisticated biological program comprised of both pro- and anti-inflammatory arms. The pro-inflammatory response, driven by the release and subsequent signaling of multiple soluble factors known as cytokines, triggers cell activation, promotes vascular permeability and induces leukocyte recruitment, accumulation, and activation, whereas the anti-inflammatory response initiates wound healing and regenerative processes for recovery . In the The purpose of the neuroinflammatory response is to re-establish homeostasis, and it is typically self-limiting. However, when this response is not properly inactivated, ongoing neuroinflammation can result in deterioration of healthy tissue, which may severely impair physiological functioning. Aberrant microglial responses and persistent neuroinflammation have been implicated in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, as well as psychological disorders , 7. In aThe PI3K-AKT cascade is a highly conserved intracellular signaling pathway present in all eukaryotic cells, acting as a central node for transducing extracellular stimuli that involves phospholipid and protein phosphorylation of various downstream substrates to induce changes in cellular responses. This pathway orchestrates growth, motility, survival, and metabolism, as well as coordinating defense mechanisms in the immune system –20. In t.In view of the central role of microglia in establishing chronic neuroinflammation and neuropathology related to disease development, this review aims to summarize the current understanding of how PI3K-AKT signaling facilitates the microglial response, and how aberrant activity in this pathway may promote chronic neuroinflammation. We also consider how manipulation of AKT signaling can induce beneficial, anti-inflammatory responses to counteract the development of neuroinflammation; and highlight knowledge gaps as well as future directions of researchMicroglia are an integral component of the neuroimmune system and act as one of the first lines of immune defense. They are described as the “macrophages of the brain” due to their similarity in function and biomarker expression, to monocyte-derived macrophages . Under nThe concept of “M1” and “M2” polarization was initially coined to describe the ability of macrophages to alter their phenotype in vitro in response to cytokines, such as interferon-γ or interleukin (IL)-4, becoming classically activated or alternatively activated, respectively. However, more recent in vivo analyses of both macrophages and microglia in experimental rodents has revealed this distinct description no longer holds up; their responses are more heterogeneous and occur in a continuum between pro- and anti-inflammatory states, such as concurrent expression of both inflammatory markers during acute responses to TBI and lipopolysaccharide (LPS) –37.Typically, microglial activation occurs in a transient manner that is intended to protect the CNS and aid recovery, by initiating both components of the inflammatory response in a temporal and spatially restricted manner. However, sustained or uncontrolled activation can have deleterious consequences. Chronic activation of microglia can result in robust changes to their inflammatory profile, associated with the continual release of neurotoxic inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines, including, IL-1 and tumor necrosis factor (TNF)-α –40. Vari2+ ion sensitivity and influx [The potential neurotoxic consequences of chronically activated, pro-inflammatory microglia have remained a topic of intensive research in recent years, and whether skewed microglial responses can directly promote the development of neurodegenerative disease is still inconclusive. However, considerable evidence suggests that chronically activated microglia continually secrete neurotoxic molecules to sustain neuroinflammation. This is supported by histological and biochemical studies demonstrating that numerous pro-inflammatory molecules are increased in both the serum and cerebrospinal fluid in the context of neurodegenerative diseases and head injuries –52. Pro-d influx –55.Despite the seemingly prominent role of microglia in the onset of chronic neuroinflammation, the mechanisms underlying the transition towards a persistent, deleterious response are still unclear. To date, multiple factors have been speculated to induce abnormal microglial activation, including aging, peripheral infection, systemic inflammation, as well as genetic predispositions and mutations , 57. Fun2). PI3K phosphorylates PIP2 to generate the key second messenger phosphatidylinositol 3,4,5 trisphosphate P3), which recruits the intracellular signaling molecule AKT ), a serine/threonine-specific protein kinase P2 or PIP2, respectively  are a family of intracellular lipid kinases that transduce signals from cell surface receptors, such as receptor tyrosine kinases, receptors that lack intrinsic tyrosine kinase activity, and heterodimeric G protein-coupled receptors . Upon liase Fig. . This leely Fig. .Fig. 1PIAbundant evidence demonstrates a central role for the PI3K-AKT signaling pathway in health and disease. Activated AKT is able to phosphorylate and activate a range of substrates in the cytoplasm and nucleus that mediate numerous cellular functions critical for survival, proliferation, differentiation, migration, and glucose uptake and metabolism –66. In aSimilarly, in the brain, dysregulation of the PI3K-AKT signaling pathway is involved in numerous pathologies, such as neurodegenerative diseases and brain tumor metastasis , 70. HowThe role of PI3K-AKT signaling in modulating microglial activity and inflammatory responses was first observed in in vitro studies almost two decades ago. Stimulating mouse BV-2 microglial cells with the anti-inflammatory cytokine transforming growth factor-beta resulted in a reduction of PI3K-AKT-driven nitric oxide (NO) production and improvement in cell survival . ConversOn the contrary, PI3K-AKT signaling may also promote an anti-inflammatory response in microglia, whereby activation of AKT may be neuroprotective. A recent study by Bhat and colleagues found that use of an anti-inflammatory modulator that promoted AKT activity in an experimental TBI model resulted in decreased pro-inflammatory microglial activation and favorable outcomes in rodents . In addiCollectively, these studies suggest that activation of the PI3K-AKT signaling cascade in microglia within an in-vivo setting can induce both arms of the inflammatory response. However, it appears that the M1/M2-like polarization of microglia is influenced by multiple factors, and more detailed characterization of activated microglia is required.A number of essential microglial cell surface receptors utilize the PI3K-AKT signaling network, with signaling from these receptors directing microglial responses both during homeostasis and neuroinflammatory disease Table  Fig. 2)2) 73, 8–85. The Colony-stimulating factor 1 receptor (CSF-1R), also known as macrophage colony-stimulating factor receptor (M-CSFR) or CD115, is a type 1 membrane protein that is critical for the development, differentiation, survival, and function of monocytes and tissue macrophages . In the Csf1r gene ablated microglial development and proliferation, resulting in congenital absence of microglia in the developed brain, which resulted in neurological and other developmental abnormalities [Csf1r−/− mice and in mice with a loss-of-function mutation in the gene encoding CSF-1 (Csf1op/op), with Csf1op/op mice displaying pronounced postnatal olfactory deficits [Csf1op/op mice appeared to be unchanged compared to mice harboring a homozygous null mutation in Csf1r [Abnormal CSF-1R expression is thought to interfere with microglia functioning and contribute to neurodevelopmental disorders and neurodegenerative diseases . Indeed,malities . Blunteddeficits , 88. Surin Csf1r , 117. Thin Csf1r .Increased CSF-1R expression and CSF-1 levels within the brain have recently been identified as mediators of demyelination in progressive multiple sclerosis by perpetuating microglial survival and proliferation to exacerbate neuroinflammation . InhibitToll-like receptors (TLR) are a distinct class of receptors expressed on an array of innate immune cells, where they recognize conserved molecular patterns in microbes. Several subclasses of TLRs have been identified on microglia, including TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 . TLRs ca2 conversion, in turn causing AKT phosphorylation at serine 473 and leading to the recruitment of NF-κB [Most TLRs, including TLR4, signal via the cytosolic adaptor protein myeloid differentiation primary response 88 (MyD88). The binding of LPS to the TLR4 complex leads to the tyrosine phosphorylation of MyD88, which induces recruitment of PI3K via its p85 domain leading to its subsequent activation Fig.  123, 12. The exaof NF-κB .Several studies have provided evidence that activation of microglia through TLR4 occurs via the PI3K-AKT pathway , 125. FoAbnormal TLR4 expression has been implicated in impaired microglial functioning and neurodegenerative diseases, presumably via its link to PI3K activity. Endogenous TLR4 expression is thought to be neuroprotective in some contexts; for example, by mediating microglial autophagy of neurotoxic plaques in primary mouse microglia and experimental rat models of Parkinson’s disease , 128. CoTriggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a transmembrane receptor of the immunoglobulin superfamily that was initially identified in monocyte-derived dendritic cells and mouse macrophages . TREM2 cTrem2 mutations with microglial disorders and disease progression [Emerging evidence has suggested that abnormal TREM2 expression and signaling within the brain may promote progressive dementia, such as Alzheimer’s disease and Nasu–Hokola disease, which is a genetic disorder characterized by early-onset dementia . Genome gression –103. Intgression . Similargression . Recentlgression , 142. Gegression , 142. Algression . TherefoCx3cr1 is one of the most highly expressed genes in human and mouse microglia, and activation of CX3CR1 on microglia induces the PI3K-AKT signaling cascade in a dose-dependent manner [CX3CR1, also known as fractalkine receptor or G-protein-coupled receptor 13, is a membrane-bound chemokine receptor expressed on most early myeloid lineage cells, which mediates their adhesion and migration in response to CX3CL1, which is mostly expressed by neurons . In the t manner , 146. Sit manner .Increasing evidence indicates that disruption to the CX3CR1 signaling pathway can produce disease-specific microglial responses that promote neuropathology in various brain disorders. Perhaps unexpectedly, an initial study reported that ablation of CX3CR1 signaling in microglia led to an exacerbated rather than attenuated microglial response, significant neurotoxicity to a peripheral LPS challenge, as well as worsened neurodegeneration in experimental models of Parkinson’s disease and amyotrophic lateral sclerosis . These eOverall, numerous studies have shown that PI3K-AKT is a key signaling pathway utilized by microglia for regulating their response to various extracellular stimuli. This pathway not only underpins microglial development and activation, but is central for maintaining brain homeostasis. Abnormal PI3K-AKT signaling as a result of dysregulation in specific cell surface receptor expression or functionality disrupts microglial activities thereby promoting neurological disease.Based on the demonstrated role of PI3K-AKT signaling in the regulation of microglial responses and inflammatory phenotype, it is safe to hypothesize that disruption to this pathway promotes chronic microglia-driven neuroinflammation and neurodegeneration  variants of the SHIP-1 encoding gene, n humans –153, 164n humans , 165. Inelopment , 155. Fuelopment . Therefoelopment . CollectPINK1 gene are linked to the recessively inherited form of Parkinson’s disease [Pink1−/− mice, likely in response to debris from neurons undergoing mitophagy [Pink1−/− mouse astrocyte and microglia cultures exhibited elevated NO production and TNF-α and IL-1β transcripts in response to LPS, IFN-γ and alpha-synuclein proteins, relative to primary glial cells from wild type mice [Similarly, dysregulation of PI3K-AKT signaling has also been linked to the development of Parkinson’s disease. Abnormal expression of the PTEN-induced serine/threonine–protein kinase 1 (PINK-1) or mutations in the  disease , 161. As disease . Increasitophagy . More reitophagy , 170. Inype mice . The undype mice . Howeverype mice . Therefoype mice , 172.Akt1 has been documented in monocytes generated from experimental disease models prior to disease development and during neurodegeneration, therefore, correlating changes in AKT activity to disease onset [The PI3K-AKT signaling pathway has also been implicated in the etiology of Huntington’s disease. Activation of this pathway in neurons via insulin growth factor 1 is protective against huntingtin-induced toxicity in experimental Huntington’s disease models; however, contrasting observations were made in the peripheral immune cell compartment of Huntington’s disease patients . Post-mose onset , 157. Inse onset , 175. Itse onset . Nonethese onset . Thus, aHowever, the current paradigm of PI3K-AKT signaling in neurodegenerative diseases is primarily based on in vitro and ex vivo work using both microglia and macrophages, and experimental rodent models. Therefore, further clinical studies of microglial responses in neurological disease settings are required to provide greater insight into how alterations in PI3K-AKT signaling induces a pathogenic state in microglia..With evidence mounting in support of a central role for PI3K-AKT signaling in neurodegenerative disease, it is feasible that modulation of this pathway may be a means to control disease pathogenesis. The readers are directed to an excellent review that summarizes specific compounds that have been utilized to modulate PI3K activity in the brain . To dateTnf-α, Il-1β and Il-6 in the injured brain and induced a doubling of anti-inflammatory factors during the first 48 h after injury, while neuronal damage was halved compared to non-treated stroke animals [Targeting PI3K in in vivo disease and injury models has likewise revealed promising neuroprotective effects. Inhibition of PI3K activity in 3–6-month-old mice in an Alzheimer’s disease model dampened microglial secretion of TNF-α, and consequently increased survival and prevented cognitive decline in comparison to non-treated mice . In addi animals . In othe animals , 183. In animals . TogetheWhile there is strong support to indicate that PI3K inhibition may be a therapeutic approach in certain neurological disease settings, there is also evidence to suggest that induction of PI3K-AKT signaling, through the use of anti-inflammatory factors, may be required in some disease contexts. The upregulation of PI3K-AKT signaling following exogenous application of the anti-inflammatory mediator fibroblast growth factor 10 in mice following experimental spinal cord injury, reduced IL-6 and TNF-α production and promoted an anti-inflammatory skewed microglial response . In anotTaken together, promising research demonstrates that the PI3K-AKT pathway can be manipulated to exert neuroprotection within various disease contexts. More specifically, the pharmacological manipulation of the PI3K-AKT signaling axis can yield neuroprotective effects, further suggesting that this pathway may be a viable target for attenuating chronic neuroinflammation.The presence of chronically activated microglia within the brain is a major contributor to sustained neuroinflammation, which can amplify pathology and thereby exacerbate disease development in a range of contexts. To date, while the signaling cascades involved in derailing the microglial response are not fully defined, the PI3K-AKT signaling pathway is increasingly recognized as a key contributor to microglial activation and functioning; and thus, maladaptation of this pathway is highly implicated in this pathogenic response. Although the importance of PI3K-AKT signaling is well established in the systemic immune system, our current understanding of PI3K-AKT signaling in the neuroimmunological context remains incomplete.This review has provided an up-to-date overview of the role of PI3K-AKT signaling in regulating microglial responses. The PI3K-AKT pathway appears to regulate both the pro- and anti-inflammatory response, with the current paradigm indicating that it serves a prominent role in initiating the production of pro-inflammatory mediators in microglia after stimulation. It then follows that, abnormal PI3K-AKT signaling in the brain can potentially derail the microglial response, promoting neurotoxicity and persistent neuroinflammation. Aging has been shown to alter PI3K-AKT signaling and dysregulation of this pathway in the brain is considered a risk factor for neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. Whether other biological factors, such as sex—which has previously been shown to alter microglial maturation, activity and responses—can influence PI3K-AKT signaling, remains unclear .Promising pharmacological studies targeting different components of the PI3K-AKT pathway suggest that this highly complex signaling cascade can be manipulated to skew the microglial response towards neuroprotection. However, further investigation into this pathway in the context of chronic neuroinflammation is needed to reveal its true potential as a viable therapeutic target for alleviating or preventing the onset of persistent neuroinflammation in injury, aging, and disease states while leaving the systemic immune system intact.


Uric acid (UA) transporters mediate the uptake and outflow of UA, and are greatly involved in the control of UA concentrations. Glucose transporter 9 (GLUT9), one of the UA transporters, has been confirmed to be expressed in human umbilical vein endothelial cells (HUVECs). This study aimed to characterize GLUT9’s effect on intracellular UA accumulation in HUVECs in a high-UA environment and to explore the mechanism of cellular dysfunction.HUVECs were treated with UA to establish a model of cellular dysfunction. Then, UA uptake, GLUT9 expression and endothelial nitric oxide synthase (eNOS) and reactive oxygen species (ROS) amounts were measured. UA uptake was concentration- and time-dependent, and UA treatment significantly reduced nitric oxide (NO) levels and eNOS activity. UA also upregulated pro-inflammatory molecules and GLUT9, and increased intracellular ROS amounts in HUVECs. GLUT9 knockdown reduced UA uptake and ROS content, but antioxidant treatment did not reduce GLUT9 expression. To assess the function of JAK2/STAT3 signaling, HUVECs were treated with UA, and the phosphorylation levels of JAK2, STAT3, IL-6 and SOCS3 were increased by a high concentration of UA. In addition, GLUT9 knockdown reduced the phosphorylation of JAK2/STAT3 intermediates and increased p-eNOS amounts.GLUT9 mediated the effects of high UA levels on HUVECs by increasing the cellular uptake of UA, activating JAK2/STAT3 signaling, and reduced the production of active eNOS and NO in HUVECs.The online version contains supplementary material available at 10.1007/s11033-021-06840-w. Uric acid (UA) constitutes the end-product of primates’ metabolic reactions transforming purine nucleosides. Although at normal concentrations, UA plays antioxidative roles in humans, epidemiological evidence indicates abnormally high UA concentrations are associated with cardiovascular abnormalities, such as hypertension, coronary heart disease, and atherosclerosis –4. HowevSerum UA concentration mainly depends on the dynamic balance between UA production and its excretion. UA transporters, including URAT1, GLUT9 and breast cancer resistance protein (BCRP), mediate UA uptake and outflow, greatly regulating UA concentrations in the body . It was 2. UA  was dissolved in serum-free F12-K medium, incubated at 37 °C with constant shaking, and filtered using 0.22-μm syringe filters  [HUVECs provided by the China Center for Type Culture Collection  underwent culture in F12-K basal medium supplemented with 10% FBS, ECGS and 0.1 mg/ml heparin sodium at 37 °C under 5% COre, USA) . The UA HUVECs were cultured in 96-well plates and administered diverse amounts of UA  at ~ 80% confluence. Cell Counting Kit-8  was utilized to assess cell viability as directed by the manufacturer.UA uptake assays were performed in serum-free F12-K medium containing various amounts of UA . A Uric Acid Assay Kit (Sigma) was then utilized for the measurement of UA amounts in the cell culture supernatant after incubation for 24 h and 48 h, as directed by the manufacturer. Next, cell lysis was performed after cell counting with a hemocytometer. UA uptake was quantified by normalizing UA concentration to the cell count.NO release was measured in the cell culture supernatant using a Total Nitric Oxide Assay Kit , as directed by the manufacturer. This was achieved by measuring nitrite and nitrate amounts with the Griess reagent. Absorbance was measured on a microplate reader at 520 nm after color development.The concentration of eNOS in the cell culture supernatant was assessed with a Human eNOS ELISA kit (Beyotime Biotech), as directed by the manufacturer. Briefly, eNOS in the supernatant was bound by an anti-eNOS antibody in an ELISA plate, and then horseradish peroxidase-labeled avidin and the developing agent were added in sequence. Absorbance at 520 nm was used to calculate eNOS concentration.4 cells/ml, stimulated with UA. The culture medium was replaced with DCFH-DA diluted in serum-free medium to 10 µM, followed by a 20-min incubation. After three washes with serum-free medium, fluorescence was immediately assessed on a Leica fluorescence microscope , with excitation and emission at 488 nm and 525 nm, respectively.Intracellular ROS concentration was assessed with DCFH-DA (Sigma). The cells underwent seeding in 35-mm plates at 4 × 10RNA extraction utilized Transzol reagent , and RNA purity was determined by absorbance measurements at 260 and 280 nm. Reverse transcription was carried out with a Reverse Transcriptase kit (TransGen Biotech), and transcript abundance was quantitated on a 7500 Real-Time PCR System  using the SYBR Green detection system (TransGen Biotech).Gapdh mRNA amounts. The 2−ΔΔCt method was utilized for data analysis. The primers used are listed in Table S1.Target gene expression was normalized to Lysates were prepared from HUVECs in the RIPA buffer supplemented with protease and phosphatase inhibitors . The BCA kit (Thermo Fisher Scientific) was utilized for protein quantitation. Equal amounts of total protein underwent separation by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), followed by electro-transfer unto PVDF membranes . Upon blocking with 5% skim milk (2 h at ambient), successive incubations were carried out with primary (overnight at 4 °C) and secondary (50 min at ambient) antibodies. After washing, detection of immunoreactive bands utilized the enhanced chemiluminescence reagent (Thermo Fisher Scientific). ImageJ was used for imaging and quantitation. Target protein expression was normalized to that of GAPDH. The utilized antibodies, provided by Abcam (UK), Proteintech (USA) or Cell Signaling (USA), were directed against GLUT9 (1:1000), eNOS (1:1000), phosphorylated (p)-eNOS (1:1000), total (t)-JAK2 (1:1500), p-JAK2 (1:1000), t-STAT3 (1:1500), p-STAT3 (1:1000), IL-6 (1:2000), SOCS3 (1:2000) and GAPDH .HUVECs underwent fixation with 4% formalin in PBS and permeabilization with 0.3% Triton X-100 for 10 min. Upon blocking with 3% BSA for 30 min, incubation was carried out with mouse anti-p-eNOS (1:500) and rabbit anti-GLUT9 (1:500) antibodies, respectively, overnight at 4 °C in a humidified environment. After washing, the cells were covered with secondary antibodies and incubated at ambient for 50 min. DAPI counterstaining was performed, and cells were finally examined under a Leica fluorescence microscope (Leica).N-acetylcysteine , an antioxidant, was supplemented to some wells for determining the impact of oxidative stress on GLUT9 expression.Small interfering (si)RNAs targeting SLC2A9 (GLUT9), shown in Table S2, were designed and produced by GenePharma (China). A 20-μM siRNA stock solution was prepared as directed by the manufacturer. Then, the transfection complex was prepared by mixing Opti-MEM , siRNA stock solution (5 µl) and siRNA-Mate transfection reagent . HUVECs were transfected using these transfection complexes with siRNA concentration of 50 nM in a 6-well plate at 37 °C for 24 h. Next, UA was supplemented to appropriate wells, and cells were subsequently collected to measure transfection efficiency by RT-PCR and immunoblot. t-test and one-way ANOVA for normally distributed data. Otherwise, a non-parametric test was applied. P < 0.05 was deemed to reflect statistical significance.SPSS 22.0  was utilized for data analysis. Data are mean ± SD. Group pairs and multiple groups were compared by Student’s UA at 5, 10 and 15 mg/dl had no clear effect on cell survival, but decreased survival was detected at a UA concentration of 20 mg/dl   Fig. D. Next, 05) Fig. E. While 05) Fig. F, G.Fig.JAK2/STAT3 signaling activation is known to worsen cellular oxidative stress. Therefore, we aimed to determine whether UA increases intracellular ROS content by inducing JAK2/STAT3 signaling, subsequently leading to endothelial cell dysfunction and inflammation. The results showed that UA treatment had no effects on JAK2 and STAT3 mRNA amounts, but SOCS3 was significantly upregulated by UA at concentrations of 10, 15 and 20 mg/dl Fig. A–C. FurtMultiple mechanisms explaining UA’s role in endothelial dysfunction and cardiovascular diseases have been suggested. It was previously demonstrated UA increases calcium amounts in the mitochondria, which leads to greater production of mitochondrial superoxide, mitochondrial dysfunction, ROS generation , 14, lowHyperuricemia was associated with lower plasma membrane expression of the UA efflux transporter BCRP, which leads to decreased UA efflux and intracellular UA accumulation . In addiNO release by endothelial cells plays a critical role in vascular homeostasis , 18. In To explore the mechanism underpinning this effect, we examined GLUT9’s role. GLUT9’s effect on oxidative stress was determined by treating HUVECs with siRNA-GLUT9 and the antioxidant NAC, respectively. The results showed that siRNA and NAC treatment reduced intracellular ROS content, and GLUT9 expression was not inhibited when oxidative stress was relieved by NAC administration. Therefore, we can speculate that overexpression of GLUT9 would increase the accumulation of UA in cells and stimulate oxidative stress, which has never been addressed previously.Inflammation is critical in initiating and sustaining vascular injury. Previous studies have shown that UA causes inflammation in endothelial cells, which manifests as upregulation and enhanced release of pro-inflammatory molecules, including IL-6, THF-α, IL-1β and MCP-1 . HoweverHigh UA levels induce endothelial cell dysfunction, which is characterized by decreased eNOS activity and NO production. These effects require GLUT9, which mediates the accumulation of UA in cells, which in turn activates the JAK2/STAT3 pathway, inducing inflammation and oxidative stress.Supplementary file1 (PDF 102 kb)Supplementary file2 (PDF 80 kb)Below is the link to the electronic supplementary material.