Synechococcus were negatively affected by OA. Moreover, the abundance of very high fluorescence viruses infecting small haptophytes tended to be lower under acidification while their putative hosts' abundance was enhanced, suggesting a direct and negative effect of OA on viral–host interactions. In the oligotrophic summer situation, we found a stimulating effect of OA on total viral abundance and the viral populations, suggesting a cascading effect of the elevated pCO2 stimulating autotrophic and heterotrophic production. In winter, viral lysis accounted for 30 ± 16% of the loss of bacterial standing stock per day (VMMBSS) under increased pCO2 compared to 53 ± 35% in the control treatments, without effects of nutrient additions while in summer, OA had no significant effects on VMMBSS . We found that phage production and resulting organic carbon release rates significantly reduced under OA in the nutrient replete winter situation, but it was also observed that high nutrient loads lowered the negative effect of OA on viral lysis, suggesting an antagonistic interplay between these two major global ocean stressors in the Anthropocene. In summer, however, viral-mediated carbon release rates were lower and not affected by lowered pH. Eutrophication consistently stimulated viral production regardless of the season or initial conditions. Given the relevant role of viruses for marine carbon cycling and the biological carbon pump, these two anthropogenic stressors may modulate carbon fluxes through their effect on viruses at the base of the pelagic food web in a future global change scenario.Anthropogenic carbon emissions are causing changes in seawater carbonate chemistry including a decline in the pH of the oceans. While its aftermath for calcifying microbes has been widely studied, the effect of ocean acidification (OA) on marine viruses and their microbial hosts is controversial, and even more in combination with another anthropogenic stressor, i.e., human-induced nutrient loads. In this study, two mesocosm acidification experiments with Mediterranean waters from different seasons revealed distinct effects of OA on viruses and viral-mediated prokaryotic mortality depending on the trophic state and the successional stage of the plankton community. In the winter bloom situation, low fluorescence viruses, the most abundant virus-like particle (VLP) subpopulation comprising mostly bacteriophages, were negatively affected by lowered pH with nutrient addition, while the bacterial host abundance was stimulated. High fluorescence viruses, containing cyanophages, were stimulated by OA regardless of the nutrient conditions, while cyanobacteria of the genus  This was done every morning in order to maintain the levels of pH in the acidified tanks at around 0.25–0.30 pH units lower than the controls (KB and NB), which were bubbled with equivalent amounts of pure air to ensure similar turbulent conditions. The lowering in pH of the acidified treatments vs. the controls is equivalent to the values projected for the end of the twenty-first century following relatively pessimistic scenarios  before the lights were turned on, on February 18 and July 7, for experiments WINTER and SUMMER, respectively (corresponding to day 1). In KA and NA, pH was artificially lowered by bubbling, in a controlled way, small amounts of COSamples for inorganic nutrients were kept frozen at −20°C until analysis, which were performed using a CFA Bran+Luebbe autoanalyzer following the methods described by Hansen and Koroleff .a (chl a) analysis according to Yentsch and Menzel  following Marie et al.  were discriminated and enumerated according to their light scatter and specific autofluorescence properties.For picophytoplankton, fresh, unstained samples were analyzed in a Becton Dickinson FACSCalibur flow cytometer at high speed , samples (1 ml) were fixed with 0.2-μm-filtered glutaraldehyde , incubated at 4°C for 15–30 min, and subsequently frozen in liquid nitrogen and stored at −80°C. Upon thawing, viruses were stained with SYBR Green I (Molecular Probes Inc.) for 10 min in the dark at 80°C and quantified after dilution with TE buffer  using flow cytometry and an optimized protocol  was estimated from in situ temperature for 3 h. After incubation, the samples were analyzed with a FACSCalibur flow cytometer as described in detail elsewhere  labeling of highly active cells , the fraction of infected cells (FIC), induced viral production from lysogens (VPI), and the fraction of lysogenic cells (FLC) were estimated for selected samples using the virus reduction approach . An increase in viral abundance in the MC treatments represents VPL + VPI  was estimated following the approach of Wells and Deming . Carbon release rates through viral lysis were calculated based on VP by converting the number of lysed bacteria into carbon using a factor of 20 fg C cell−1  with time as a covariate was used to discern the effects of time from those of the lowered pH and nutrient amendment for time-averaged parameters. Kruskal–Wallis tests for non-normal distributions were used to evaluate the acidification effect for K and N treatments, separately. Spearman rank correlation for non-parametric data was performed to determine the relationships between the various measured parameters.All statistical analyses were performed with JMP 7.0 (SAS). The Shapiro–Wilk in situ nutrient concentrations (phosphate and silicate) were four to five times higher than in SUMMER. Initial experimental nitrogen concentrations in the N treatments were higher in the WINTER than in the SUMMER experiment, as they were added by multiplying the monthly average concentration measured in the BBMO during the last 10 years by a factor of 8  than in the SUMMER experiment  and increased significantly in the nutrient-amended treatments with time and with respect to the controls  doubled that observed in SUMMER  and increased significantly with nutrient additions in both experiments , increased with nutrient addition, and was stimulated by the lowered pH only in SUMMER  than in the SUMMER experiment , and BA dynamics differed between seasons: in WINTER, BA increased 7.6 times from day 0 until day 4 without differences between treatments .Total VLP abundance increased after an initial stable phase from day 7 and 5 in experiments WINTER and SUMMER, respectively, and the same behavior was exhibited by the low subpopulation ; this inn = 24, P < 0.05). Total VLP and the subpopulation of low fluorescence VLP, i.e., mostly bacteriophages, were negatively affected by lowered pH with nutrient addition . In the nutrient treatments, the medium fluorescence subpopulation abundances, also comprising bacteriophages, were triggered in the acidified containers for the time interval 5–7 days (P = 0.0011).In the nutrient-deplete SUMMER experiment, in turn, total VLP abundance and the abundances of all subpopulations were correlated negatively to the measured pH in the mesocosms , i.e., iL) did not differ significantly between seasons and averaged 0.68 ± 0.32 × 107 VLP ml−1 day−1 but evolved differently between experiments  with respect to KB . VPL was significantly stimulated by nutrient addition  with a trend of reduced VPL due to lowered pH . Lysogenic production could only be induced at day 0 (0.69 ± 0.01 × 107 VLP ml−1 day−1) and in the NA treatments at day 5  and were not affected by lowered pH. In the NB treatments, lysogenic production rates accounted for 100% of virus production, i.e., virus production could only be detected in the treatments where lysogens were induced; VPI were 0.30 ± 0.11 × 107 and 0.48 ± 0.13 × 107 VLP ml−1 day−1 at days 4 and 8, respectively, being lower or equal compared to the VPL rates in the acidified, i.e., NA, treatments. Moreover, VPI could only be induced at day 0 without nutrient addition (0.33 ± 0.11 × 107 VLP ml−1 day−1) and at day 4 in the KA treatments  and was not affected by acidification. The FLC also remained low throughout the experiments and treatments without discernable treatment effects  and 16 ± 10 (4–43) in SUMMER. The fraction of infected bacteria (FIC) was low in both experiments and all treatments and accounted for 13.9 ± 18.9% in WINTER and 4.8 ± 3.2% in SUMMER. Only in WINTER, FIC decreased with nutrient additions  did not differ between seasons (39 ± 28% in WINTER and 41 ± 22% in SUMMER) but was initially lower in WINTER (21 ± 5%) than in SUMMER . Carbon released from the particulate to the dissolved pool due to viral activity was four times higher in WINTER (15.0 ± 5.2 μg C L−1 day−1) than in SUMMER  and also stimulated by nutrient addition .The viral-mediated loss of bacterial standing stock  and was positively correlated to chl a concentration and BA . However, when separating by seasons and nutrient treatments, the trend of increasing viral production and viral-mediated mortality with increasing pH was significant only in the K treatments of the WINTER experiment  coupled to increased host production under elevated pCO2, suggesting that lower viral production was not a secondary effect of reduced host production but a direct and negative effect on viral–host interactions. Maat et al.  and to Prochlorococcus  lends further support that this group was mainly comprised of cyanophages. Thus, the negative response of Synechococcus in WINTER could be the consequence of enhanced viral lysis in this season. Indeed, freshwater cyanophages have been shown to tolerate a broad range of pH  gen amplicons from the present study showed the synergistic effects of OA and nutrient amendment selected for some specific OTUs belonging to SAR86 . There, bacterial production was either not affected , recurrently accumulate in the stratified, oligotrophic conditions of early summer (Ortega-Retuerta et al., Instead, phage-mediated loss of bacterial biomass in WINTER was apparently the dominant mortality agent for bacteria there. Since bacterial abundance was higher in WINTER than in SUMMER, this may have increased the encounter probability with the host. In addition, it has been suggested that viruses are the prevailing mortality agent in eutrophic waters over protistan grazing (Steward et al., a ratio estimated for bloom conditions in the Northwest Mediterranean (Gutiérrez-Rodríguez et al., We found phage production and concomitant organic carbon release rates significantly reduced in the acidified treatments of the nutrient replete WINTER experiment, especially in the non-amended treatments. When comparing the organic carbon processed by the viral shunt with the total autotrophic biomass converted to organic carbon with a C/Chl pCO2 concentrations has often been observed in published studies (Kim et al., While no significant differences in the net accumulation of DOC and the protein-like DOM compounds could be detected between the acidified and non-acidified conditions in the present study (Aparicio et al., pCO2 led to greater DOC removal by bacteria growing on a range of added DOC types, from glucose to complex phytoplankton products (James et al., pCO2. Reduced viral lysis under OA, however, should counterbalance these negative effects of lowered pH on the biological carbon pump, at least in bloom and postbloom situations where viral lysis plays a relevant role in carbon cycling through the microbial food web.Seawater culture experiments with bacterioplankton communities from contrasting sites such as the coastal upwelling influenced Santa Barbara Channel, and the oligotrophic systems of the Sargasso Sea and the South Pacific Subtropical Gyre evidenced that elevated pCO2. In the oligotrophic SUMMER situation, where viral-mediated carbon fluxes were less important compared to WINTER, OA stimulated viruses through the cascading effect of elevated pCO2 on the autotrophic and heterotrophic production. Given the relevant role of viruses in marine carbon cycling and the biological carbon pump, these two anthropogenic stressors may modulate carbon fluxes through their effect on viruses at the base of the pelagic food web in future global change scenarios.Our experimental results provide insight into how two major global change pressures, ocean acidification and eutrophication, modulate viral production and viral-mediated processes in the plankton community at different seasons. While nutrient loading consistently enhanced phage production regardless of the season, the effect of OA on viruses differed between seasons. In the nutrient-replete bloom/postbloom situation in WINTER, viral production and phage-mediated bacterial mortality were reduced under increased pCO2 and nutrient additions simulating the combined effect of acidification and eutrophication. This work contributes to the understanding of how these two anthropogenic stressors modulate carbon fluxes at the base of the pelagic food web in future global change scenarios.This is the first study that shows phage-mediated bacterial mortality leading to altered carbon fluxes in response to elevated The original contributions presented in the study are included in the article/AM, JB, VB, EC, CP, JP, JG, CM, MS, and DV conceived, planned, and carried out the mesocosm experiments. JB and AM performed the mortality experiments. AM analyzed viruses by flow cytometry and took the lead in writing the manuscript. All authors provided critical feedback and helped to shape the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mesenchymal stem cells are a continually expanding area in research and clinical applications. Their usefulness and capacity to differentiate into various cells, particularly neural types, has driven the research area for several years. Neural differentiation has considerable usefulness. There are several successful differentiation techniques of mesenchymal stem cells that employ the use of small molecules, growth factors and commercially available kits and supplements. Phenotyping, molecular biology, genomics and proteomics investigation revealed a wealth of data about these cells during neurogenic differentiation. However, there remain large gaps in the knowledge base, particularly related to cytokines and how their role, drive mechanisms and the downstream signalling processes change with their varied expression throughout the differentiation process. In this study, adult mesenchymal stem cells were induced with neurogenic differentiation media, the cellular changes monitored by live-cell microscopy and the changes in cytokine expression in the intracellular region, secretion into the media and in the extracellular vesicle cargo were examined and analysed bioinformatically. Through this analysis, the up-regulation of key cytokines was revealed, and several neuroprotective and neurotrophic roles were displayed. Statistically significant molecules IFN-G, IL1B, IL6, TNF-A, have roles in astrocyte development. Furthermore, the cytokine bioinformatics suggests the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is upregulated, supporting differentiation toward an astroglial lineage. Mesenchymal stem cells (MSCs) have been an in-vogue research topic for more than a decade ,2. Due tNeural differentiation can be initiated in MSCs by a range of published methods providing a range of progeny cells dependent on the concentration and timeframes used ,9. Some Cytokines are pleiotropic signalling molecules produced by almost all cells in the body that assist cell to cell communication . They arCytokines play critical roles in the developing brain and central nervous system, promoting cellular migration, maturation and differentiation into specific cell types such as neurons and glial cells ,28,29. TAs such, the aim of this study was to comprehensively characterise MSC cytokines as they undergo neural differentiation. The primary adipose-derived stem cells (ADSCs) were treated with two commercial media formulations, neurobasal media B27 and terminal differentiation media (TDM), and examined the cytokine expression changes between the undifferentiated and the treated cells. Furthermore, the secreted material and the extracellular vesicles were also isolated from each cell type for a comprehensive investigation. Using live cell microscopy, the morphological changes were tracked through temporal differentiation selected timepoints. The cytokines and chemokine analysis were completed using a sandwich ELISA multiplex analysis to quantify 27 separate cytokines in each sample type. The post hoc analysis investigated the roles the measured molecules have in signalling cascades and ontological analyses to gain insights into their role in neural differentiation.(A)The characteristic semi-fibroblastic morphology of the control ADSCs with short tripolar regions and uniform cytoplasmic regions is presented. The overall population grows in an irregular pattern across the culture vessel;(B)Control ADSCs grown for 28 days in the control media are presented. This shows a unique view of the ADSCs in the Day 0 control compared to the day 28 B27 and TDM cultures. The ADSCs at day 28 appear to be highly stressed with overgrowth, cell death and areas of senescence;(C)ADSCs treated with the B27 media additive at 14 days post-treatment show a marked difference from the controls, with cells exhibiting the elongation of cells with bipolar region extensions reaching between cells. The population displays a somewhat irregular growth pattern with some directional polarity appearing in the righthand side of the image;(D)The ADSCs 28 days post B27 treatment are presented with a vastly different population compared to the controls, where the majority of the cells have now adopted the elongated bipolar shape with a dense central body. The cells now extend between neighbouring cells. In addition, the population have adopted a much more directional growth pattern resembling fingerprint rivers and bifurcations;(E)TDM treatment in continuous culture at day 14 shows relatively similar changes as the B27, with cell projections and elongation with fine pseudopodia-like structures with extension formation;(F)Post-TDM treatment, the cells show loosely directional growth such as the B27 at day 14; the cellular morphology is also a bipolar shape; however, the cells appear to be wider and more diffused, with the central body being flatter and wider than the B27 at 28 days;(G)The cell count from the control ADSCs and the final timepoints for B27 and TDM treated ADSCs are presented. Day 14 B27, TDM and day-28 control ADSCs were not performed as the former were in a continuous culture and the latter were not useful for any further analysis. The B27 and TDM treated ADSCs show a statistically significant increase in cell number compared to the controls.In g. Cytokine levels from each sample were analysed using the Bioplex 27-plex human pro-inflammatory kit to quantitatively measure 27 distinct cytokines simultaneously  for group clustering and interaction pathway process analysis . GraphinMesenchymal stem cells (MSCs) were a substantial topic of research in a variety of areas during the last decade ,7,30. ThLive microscopy viewing of cellular morphology during temporal differentiation provides a direct comparison of cells over time in various treatment regimens, allowing the identification of key features and visual checks for overall cellular health and differentiation status . The staThe mounting evidence of MSCs capability to differentiate toward neural-like lineage is substantial with a thematic link of an assisted or directed induction with a stimulus media. Commercial differentiation media kits and formulations boast a wide user base with a varied range of stem cell types. Gibco’s B27-supplement in neurobasal media was initially intended as a neural cell support media; the literature indicates its comprehensive use in neural differentiation in embryonic stem cells ,42, iPSCIL-13′s defined classical function as a regulator of Th2 cells mediates its production of IgE . HoweverThe expression of PDGF-bb in the B27 treatment is approximately double that measured in the controls; furthermore, the TDM treatment also shows an approximate 40% increase in expression. PDGF-bb function, as a growth factor, has varied roles, with it being most prominently known for development, cell proliferation, migration and angiogenesis . SimilarNeurotrophic and neuroprotective roles also extend to IL-15 with elevated intracellular levels which were only observed in the B27 treated cells. Interestingly, the TDM final levels were lower than the control ADSCs. From an intracellular perspective, IL-15 has a substantial role in cascade activation with fulcrum modulatory effects in JAK/STAT or MAPK pathways that are vital to proliferation and differentiation. IL-15 inflammatory function also plays an important role in neuroinflammation, having a guiding role in neuroplasticity and neurogenesis . IncreasThe remaining molecules, MCP-1, RANTES and VEGF, have an increased expression in only the B27 treated cells. The literature indicates that mature neural cells utilise these factors in the neuroprotective route as well as to promote the maturation of specific neural cell types. MCP-1 are noted to have the associated neuroprotective effects when directly expressed in perturbed astrocytes and activated microglia ,64. The Cellular communication between cells is an intricate network of molecules that are transferred to immediate neighbours and those that are shuttled further afield across tissues, organs or systemically. This is achieved either by active, passive or assisted travel for a range of molecules that can be secreted directly to the extracellular space or packaged as cargo within extracellular vesicles (EVs) to conduct the array of molecular crosstalk between cells. In this study, both EVs and secreted material produced directly from cells into surrounding media were examined in parallel to the intracellular cytokine content.The measured EV cytokines are an abundant collection with noticeable clusters of growth factors, chemokines and some subsets of interleukins across the control ADSCs. Post B27 and TDM treatments show a sizeable decline across the entire panel. The standout clusters with concentrations dropping to the lowest detectable range are IL-13, MIP-1a, GM-CSF, IL-1b, IL-10, IL-4, MIP-1b, IL-12, IL-15 and IL-5. Interestingly the cytokines measured from the B27 sample EVs have less downregulation compared to the TDM samples, except for Eotaxin. Eotaxin’s final concentration in the TDM samples is more than double that seen in the B27 samples. Interestingly this trend is matched in the secretion samples but not in the intracellular samples. Few studies have explored the role Eotaxin has in these cells. Current indications point to a hypothesis that an increased Eotaxin levels in the brain may decrease neurogenesis and inhibit early differentiation ,73. ThusWhile not all cytokine changes are statistically significant, minor modulations in concentrations can lead to complex biological significance downstream, particularly in signalling systems during differentiation. While it is not possible to explicate all roles for each individual cytokine change, a group cluster gene ontology analysis benefits here for clarity. The gene ontology analysis provides a clear snapshot of the complexity of the various roles and dynamics cytokines play in signalling and differentiation. Exploring their roles in differentiation, the central hub node in the network was found to be ‘glial cell activation’, where several subsidiary cytokines were all upregulated. The interacting nodes hold substantial relevance for the types of differentiation and signalling that occurs during the treatment phases. The node ‘cell differentiation’ has 22 of the 27 measured cytokines involved within it. Interacting ontology nodes indicate directing the differentiation of towardspecific neural cell types of glial cells. Interestingly there were no neuronal ontologies observed.The increase in positive regulation of peptidyl-tyrosine phosphorylation ontology  is imporIn summary, this study treated ADSCs with two commercial differentiation media formulations and examined the cytokines within the cells, secretions and in EVs at the chosen differentiation endpoints comparing these to the undifferentiated control. The observed intracellular and extracellular changes across the litany of modulated cytokines supports the findings that both ADSCs treatments promote differentiation toward astrogliogenesis. This is further confirmed by direct live cell imaging, where cells have a distinct and sizeable shift in shape and structure. The molecular analysis heatmap clustering, gene ontology analysis and the interaction network topography visually summarise these comparative findings across the various sample types. The upregulation of key cytokines intracellularly promotes neuroprotective and neurotrophic factors that are integral during the stress of morphological changes imbued during differentiation. Furthermore, the cytokines and chemokines secreted from the cells may play a sizeable role in instigating a signalling shift and promoting the JAK/STAT cascade that, in turn, favours astrogliogenesis differentiation. Whilst they are small molecules, cytokines play an enormous role in the regulation, maintenance, signalling, differentiation and cellular fate. While secreted and intracellular cytokine effects are being studied extensively, the effects of transmission through extracellular vesicles are only just becoming more understood. Their pleiotropic and multifunctional nature is slowly being unravelled. The targets and effects of cytokine expression by the various transmission pathways are the subject of future research.Future studies may also investigate extended timepoints with targeted surface marker quantification to investigate the extent of differentiation and maturation achieved in this system. These studies may lay the foundations for refined translational applications toward modulating and promoting neural repair in situ for traumatic injury or degenerative disease patients.2 in triplicate (n = 3) in separate T175 flasks  in a media mixture comprised of DMEM Glutmax/F12  with 10% Foetal Bovine Serum  incubated at 37 °C at 5% CO2. ADSCs media was aspirated and replaced every 84 h, and cells were passaged 3–5 times by stripping cells with TrypLE Express (12604 Gibco) before being used in differentiation experiments. Cells were maintained to 80% prior to commencing differentiation experiments. Cells were counted with an automated cell counter, Countess 2 , as per the manufacturer’s instructions.Adult human ADSCs isolation and expansion procedures were outlined by Santos et al.  utilisinSub-confluent ADSCs were washed twice in pre-warmed sterile D-MEM/F12 ; this was then replaced with the differentiation media B27 or terminal differentiation media (TDM). The B27 media was comprised of Neurobasal media  supplemented with B27  and cultured for 28 days with media aspiration and replacement every 84 h. TDM was comprised of DMEM Glutmax/F12 , 2% B27 , 1% N2 , 20 ng/mL BDNF  and cultured for 28 days with media aspiration and replacement every 84 h. Then, cells were harvested post-EV and secretion collection by washing adhered cells in PBS then stripping with TrypLE Express (12604 Gibco). Cell counts were completed using Countess 2  according to the manufacturer’s guidelines. Harvested cells were then stored at −80 °C until sample preparation.g for 1 h at 4 °C to pellet EVs. The pellet was then resuspended in 1× sterile phosphate-buffered saline (PBS)  and centrifuged at 2000× g for 1 min to remove debris. The supernatant was centrifuged again at 22,000× g for 30 min at 4 °C to pellet EVs. The EVs were resuspended in PBS and stored at −80 °C until sample preparation. EV concentrations were determined by protein content using a Qubit protein assay  using the manufacturer’s protocol.EVs were isolated as similarly outlined in Dalla et al. , where gThe conditioned media from each cell sample was collected post EV isolation. Once EVs were pelleted, 500 μL of supernatant was collected from each sample and stored at −80 °C until sample preparation.g, then lysed to release inner cytokines using a probe sonicator  3 times with 10 s bursts each while on ice. Lysed ADSCs and EVs were then centrifuged at 20,000× g for 10 min to remove debris, collecting supernatant in fresh Eppendorfs for analysis. Secretion samples were also centrifuged at 20,000× g for 10 min, collecting supernatant in fresh Eppendorfs for analysis.All samples were retrieved from −80 °C and thawed on ice. ADSC and EV samples were prepared in the same manner were pellets stored in PBS were centrifuged for 10 s at 10,000× t-test where the p-value was less than 0.05. The concentration was normalised relative to sample cell number.Bioplex analysis was performed as per Santos et al.  using thData analysis was completed using Microsoft Excel 365, DanteR software   and Cyto


The aim of this study is to compare the endothelial cell density (ECD) and morphology between diabetic mellitus (DM) and nondiabetic patients at 1 year after phacoemulsification in operated eyes and nonoperated eyes.Evaluation was performed in 28 patients (56 eyes) with type 2 diabetes and 37 patients (74 eyes) without diabetes who underwent 1-year interval cataract surgery. Using a noncontact specular microscope and Scheimpflug rotating camera, corneal parameters were analyzed before and 1 year after surgery. Subgroups analysis was performed based on a disease duration 10 years and HbA1c concentration 7% and Pearson correlation analysis was performed.r = −0.474, P = .011).The mean change in ECD at 1 year after surgery was 13.28% in the DM group and 11.40% in the control group. In the fellow nonoperated eyes, the mean change was 4.47% and 3.63% in the DM and control groups, respectively. There was no significant difference in postoperative ECD, coefficient of variance, hexagonality, and central corneal thickness between 2 groups. In the subgroup analysis, the long disease duration DM group (≥10 years) had a significantly greater ECD loss than the control and short disease duration DM groups (<10 years). Blood urea nitrogen (BUN) showed a significant correlation with postoperative ECD change (The diabetic group with a longer disease duration showed significantly greater ECD decrease compared to the nondiabetic group and BUN correlated with ECD changes after phacoemulsification. Postoperative ECD loss may be high if the disease duration is long or if the BUN level is high. Therefore, corneal endothelial cell damage remains a major concern after phacoemulsification. Since the corneal endothelial cell density (ECD) is known to decrease with age, endothelial cell damage can be significant in older people. In addition to age, systemic diseases such as diabetes can also affect the corneal endothelium.Cataract is the leading cause of blindness worldwide. Diabetes also decreases Na+K+-ATPase activity in endothelial cells that play a role in maintaining the endothelial structure. These mechanisms can reduce corneal endothelial cell function.The mechanism by which diabetes causes corneal endothelial cell damage has not been clearly identified, but several hypotheses have been proposed. In diabetes, oxidative stress can occur due to the accumulation of advanced glycation end products in corneal endothelial cells.–26 According to the reported results, after cataract surgery, the loss of ECD in diabetic patients was 5.95% to 29.07%, compared to 0.88% to 18.18% in healthy individuals.–17 Previous studies have shown varying results on whether diabetic patients have an increased risk of ECD loss after phacoemulsification. In addition, most of the studies evaluated short-term changes within 3 months, and there are few papers analyzing the effect of diabetic duration and results from serum laboratory tests such as HbA1c and blood urea nitrogen (BUN) on ECD loss.,17 Since loss of corneal endothelial cells can occur naturally over time, it is important to compare both the operated and the nonoperated eyes.Several studies have shown that diabetic patients may be more susceptible to corneal complications before and after intraocular surgery.The purpose of this study was to compare the 1-year change of ECD, coefficient of variance (CV), hexagonality, central corneal thickness (CCT) in operated eyes and their nonoperated eyes in diabetic and control patients who underwent 1-year interval cataract surgery. Subgroup analysis was performed according to the diabetes duration and HbA1c concentration. Moreover, we aimed to determine the association of ECD loss with laboratory data, including ocular and systemic parameters such as disease duration, HbA1c, BUN, and serum creatinine.22.1This retrospective study included 130 eyes of 65 patients (56 diabetic and 74 nondiabetic eyes) who underwent 1-year interval phacoemulsification at the Kyung Hee University Hospital at Gangdong, Seoul, Korea, between January 2016 and December 2019 by 2 experienced surgeons using a standard technique with the same phacoemulsification machine. The inclusion criterion was grade II or III cataract according to the Lens Opacity Classification System III. Patients with a history of ocular surgery, dry eye disease, uveitis or ocular inflammation, corneal opacities, glaucoma, and ocular trauma were excluded from the study. Patients who underwent cataract surgery were divided into the diabetic mellitus (DM) and control groups, and the nonoperated were also analyzed by being dividing into the DM and control groups. The diagnosis of diabetes was based on the medical records of those under medical treatment for glucose control. We performed a subgroup analysis for diabetic patients with diabetes duration greater and less than 10 years and HbA1c greater and less than 7%. This study was approved by the Institutional Review Board of Kyung Hee University Hospital at Gangdong . Informed consent was waived because of the retrospective nature of the study and the analysis used anonymous clinical data.2.2Preoperatively, all patients underwent complete ophthalmic evaluation, including slit-lamp examination, noncontact tonometer, and posterior segment examination with pupil dilation. ECD, percentage of hexagonal cells, and CV in cell size were evaluated preoperatively and 1 year postoperatively using a Topcon SP 3000P Specular Microscope  in all patients. CCT was measured using a Scheimpflug rotating camera . Axial length and anterior chamber depth were measured using A-scan . Serum samples of patients taken before surgery were analyzed to evaluate HbA1c, BUN, and creatinine levels.2.3All procedures were performed under topical anesthesia. Phacoemulsification and implantation of an intraocular lens were performed by 2 experienced surgeons via a 2.80 mm sutureless clear corneal incision with Sovereign Compact Cataract Extraction System . The intraocular lens was placed within the capsular bag in all cases. Postoperatively, all patients received the same treatment regimen consisting of a combination of an antibiotic and a steroid.2.4t test in SPSS version 18.0 software . Comparisons of pre- and postoperative ECD, CV, hexagonality, and CCT between the 2 groups were calculated using an unpaired t test. Pearson correlation was used to analyze the correlation of ECD changes with the clinical characteristics of diabetic patients, including disease duration, HbA1c levels, serum glucose, serum urea nitrogen, and serum creatinine. A P-value of less than .05 was considered statistically significant.Statistical analysis was performed using the independent samples 3P > .05). HbA1c and blood glucose levels were significantly higher in the DM group than in the control group (P = .000). However, preoperative serum BUN and creatinine did not show statistically significant differences between the 2 groups.A total of 130 eyes of 65 patients  were included in the study: 28 patients in the DM group and 37 patients in the control group. Preoperative demographics are shown in Table The ECD in both the DM group and the control group decreased significantly compared to the preoperative ECD in the operated eye, but in the fellow non2operated eye, neither the DM nor the control group's ECD decreased significantly after 1 year , 14 eyes in the high HbA1c group (≥7%), 15 eyes in the short DM duration (<10 years) group, and 13 eyes in the long DM duration group (≥10 years) were included. In all 4 subgroups, postoperative ECD was significantly reduced compared to preoperative ECD   Table .4,27,28 This implies that diabetes is a potential risk factor for ECD loss after phacoemulsification. During cataract surgery, age, short axial length and shallow anterior chamber depth, high lens density, and large corneal incision size are factors known to affect corneal endothelial reduction.,29,30 In this study, there was no statistically significant difference in preoperative age, lens density, axial length, and anterior chamber depth between the diabetes group and the control group. In addition, preoperatively, endothelial cell parameters were similar in both groups, indicating that no sampling bias was present  than the known values. This is likely to be higher than the typical rate of loss during lifetime because the mean age of the patients included in this study was over 60 years old.In this study, unlike other previous studies, the fellow eye not receiving surgery was also evaluated in order to confirm the natural decrease in ECD. The ECD loss for 1 year in the fellow eye was 4.47% in DM patients and 3.63% in the control group, and there was no significant difference in corneal endothelial cell parameters between the 2 groups before and after surgery. In general, ECD has its highest density at birth at 7500 cells/mm reported that there was no difference between groups when DM patients were divided based on a disease duration of 10 years, and there was no association with HbA1c levels. They explained that the reason for these results was that type 2 diabetes had no impact on corneal cell density in subjects with good glycemic status. However, another study reported that DM of more than 10 years was associated with more significant natural ECD changes. This is because the longer the disease duration, the higher the probability of chronic microvascular complications due to chronic exposure to hyperglycemic conditions, and the corneal endothelium becomes vulnerable. Despite the absence of significant differences between the diabetic and control groups with respect to preoperative ECD, postoperative ECD and ECD loss, diabetic patients in the current study demonstrated more ECD loss postoperatively compared to nondiabetic patients. Therefore, diabetes duration may be considered to affect postoperative ECD loss and further studies are needed.In the subgroup analysis, the ECD loss in the long DM duration subgroup was significantly higher than that in the short DM duration subgroup and the control group. Few studies have reported ECD loss after cataract surgery according to disease duration and glucose control. Unlike the results of this study, Kudva et al,5,10,35 In addition, diabetes also impairs the activity of the Na+K+-ATPase enzyme in endothelial cells. These mechanisms can reduce the cornea's ability to control corneal hydration. There is also a report that corneal endothelial cells are easily damaged in case of the lower initial endothelial cell count.–21The large ECD loss in diabetic patients is because their endothelial cells are vulnerable to trauma caused by cataract surgery. Potential theories for increased vulnerability include oxidative stress due to chronic overall ischemia, accumulation of advanced glycation end products occurring in diabetes, and underlying corneal neuropathy, making the eye more susceptible to the damaging impact of surgery.,6,19 However, even though it was not statistically significant, the preoperative and postoperative CV values were larger in the DM group than in the control group. Surgeons should be aware that the recovery of endothelial cells may be slower and less significant in diabetics. Hexagonality indicates the variability in hexagonal cell shape, such as CV, it means the healing response after damage. In this study, hexagonality also decreased at 1 year postoperatively in the DM and control groups, but the difference was not statistically significant. Various results have been reported previously. Lee et al and Morikubo et al reported that there was no difference in hexagonality between the DM and control groups, and Hugod et al showed a significant decline in the percentage of hexagonal cells only among diabetic patients. One year is a sufficient amount of time for the corneal endothelial cells to recover, and therefore, it is believed that there was no significant difference in this study. CCT was used as a marker for endothelial function. Similar results were found in previous studies. Hugod et al and Elbassiouny et al reported that there were no differences in CCT between diabetic and nondiabetic patients after surgery. This is because there is ECD loss after cataract surgery, but this is not sufficient to affect corneal endothelial cell function.Meanwhile, CV, hexagonality, and CCT did not show statistically significant differences between the DM and control groups both before and after surgery. CV is an indicator of the uniformity of the size of endothelial cells, which indicates the activity of the repair and healing mechanism of the endothelium after damage. Previous studies demonstrated nonsignificant differences in CV changes in diabetic patients after cataract surgery.,37 Regarding renal function and corneal endothelial cell status, Ohguro et al reported that polymegethism and pleomorphism were increased in patients with chronic renal failure. The amount of urea in the aqueous humor is known to increase in proportion to blood urea, and oxidized glutathione is increased in the aqueous humor in patients with renal failure, which is described as urea, and may be toxic to the corneal endothelium.,40 However, since our sample size was small, additional research and analyses are needed.In this study, we determined a significant negative correlation between postoperative ECD loss and BUN in patients in the diabetic group. BUN is an indicator of the gross index of glomerular function. In previous studies on its effect on corneal endothelial cells, BUN was significantly higher in DM patients than in the control group, but it did not affect the corneal endothelium, and other study reported that the urinary albumin-creatinine ratio was correlated with ECD.This study is limited by its small sample size and its retrospective nature; therefore, our findings cannot be generalized to all patients who undergo phacoemulsification in diabetes. In addition, during phacoemulsification, trauma by ultrasound energy plays a significant role in ECD loss. In this study, ultrasound energy could not be analyzed. Despite these limitations, unlike previous studies, our study reported ECD change over a relatively long term of 1 year in diabetes after phacoemulsification, and through subgroup analysis, it was confirmed that ECD loss was large when the disease duration was 10 years or longer. Based on this, our results add further evidence to the literature regarding the effect of DM on ECD loss after phacoemulsification.5In conclusion, there was a statistically significant decrease in ECD 1 year after surgery in both the diabetic and control groups. In the subgroup analysis, the long-duration DM group (≥10 years) showed a significantly greater postoperative ECD loss than the control group and short-duration DM group (<10 years) and BUN showed correlation with postoperative ECD decreases. Therefore, in DM patients with a duration of 10 years or more or with high serum BUN, the possibility of ECD reduction after cataract surgery should be considered when planning cataract surgery.Conceptualization: Tae Gi Kim.Data curation: Jin-Ho Joo.Formal analysis: Jin-Ho Joo, Tae Gi Kim.Funding acquisition: Tae Gi Kim.Investigation: Tae Gi Kim.Methodology: Jin-Ho Joo, Tae Gi Kim.Writing – original draft: Jin-Ho Joo, Tae Gi Kim.Writing – review & editing: Tae Gi Kim.


We also analyzed these factors in relation to DNA methylation in saliva of stress-related candidate genes (SLC6A4 and FKBP5), generated via bisulfite pyrosequencing (complete case n's range from 67–72 baseline and 29–31 follow-up) (n's range from 80 baseline to 36 follow-up). We found various associations with cardiometabolic risk, such as higher social support and greater acculturation were associated with lower BMI in mothers; discrimination and school stress associated with greater waist circumferences in children. Very few exposures associated with FKBP5, but various stressors associated with methylation at many sites in SLC6A4, including immigrant-related stress in both mothers and children, and fear of parent deportation in children. Additionally, in the mothers, total maternal stress, health stress, and subjective social status associated with methylation at multiple sites of SLC6A4. Acculturation associated with methylation in mothers in both genes, though directions of effect varied over time. We also find DNA methylation at SLC6A4 associates with measures of adiposity and blood pressure, suggesting that methylation may be on the pathway linking stress with cardiometabolic risk. More research is needed to determine the role of these epigenetic differences in contributing to embodiment of stress across generations.Psychosocial stressors can become embodied to alter biology throughout the life course in ways that may have lasting health consequences. Immigrants are particularly vulnerable to high burdens of stress, which have heightened in the current sociopolitical climate. This study is an investigation of how immigration-related stress (IRS) may impact the cardiometabolic risk and epigenetic markers of Latinx immigrant mothers and children in Nashville, TN. We compared stress and resilience factors reported by Latina immigrant mothers and their children (aged 5–13) from two time points spanning the 2016 U.S. presidential election (June 2015–June 2016 baseline,  Many studies have identified associations between adverse childhood experiences, socioeconomic adversity, and other environmental stressors with lower DNA methylation of FKBP5 across various tissues as reviewed in Parade et al.  in saliva . This time point is referred to as “follow-up” throughout the study. Informed oral consent was provided by all participants, and Vanderbilt University and University of California San Diego Institutional Review Boards approved all protocols. All data are available upon request, and key measures are available in The study sample draws from a longitudinal study of stress embodiment, entitled “Children of Hispanic Immigrants Collaborating to Overcome Stress” (CHICOS). The study recruited 81 families (mother-child dyads) in Nashville, TN between June 2015 and June 2016. This initial time point is called “baseline” throughout the rest of the study. Participants were recruited from local immigrant-serving community centers with subsequent snowball sampling. Inclusion criteria were self-described Latina, foreign-born immigrant mothers above age 18, with a child between the ages of 5–13. The mother and focal child were assessed on a number of different biological, psychosocial, and health-related measures. Following baseline assessment of the dyads, we revisited the same families 2–3 years later (March–September 2018), collecting the same data and new interviews focused on changes since the 2016 presidential election, from all available mothers and children who participated in the baseline sample (All exposure data were collected using surveys administered in person with mothers in Spanish (1.5–2 h) and children primarily in English (45 min). Surveys were a combination of validated scales and new survey questions developed following a set of preliminary individual interviews in 2014 with children, and focus groups with Latina immigrant mothers in Nashville, TN . Questioimmigrant-related stress (IRS), discrimination stress, and a total stress score. Measures additionally reported by only mothers included family economic stress, family health stress, and household stress. A measure reported only by the children was school stress. The questions included in each stress scale and Cronbach's alpha score for internal reliability are available elsewhere  was also considered a resilience factor, as higher scores indicate better social standing. SSS has been linked with self-reported health among immigrant Latinas, and has been hypothesized to capture immigrant experiences that may alter perceived self-worth  percentile (adjusted for child age and gender) and waist circumference. In the mothers, we measured BMI, systolic blood pressure (SBP), and diastolic blood pressure (DBP).via bisulfite pyrosequencing at two CpG sites within intron 7 of the FKBP5 gene and six sites within the promoter region of the SLC6A4 gene. In the end, we generated high quality methylation data from 78 child and maternal samples at baseline and 36 child and 38 maternal samples at follow-up for FKBP5. For SLC6A4, we generated data for 78 child and 80 maternal samples at baseline and 37 samples for both groups at follow-up. These gene regions were chosen based on prior studies highlighting their importance in early life adversity in humans, other primates, and rodents , which covers two sites in Bin 2 of intron 7  using standard protocols . Genomic DNA was also extracted from saliva of 80 mothers at baseline and 40 mothers in follow-up. Saliva was stored at room temperature, per manufacturer's recommendation until DNA extraction. DNA was isolated from 500 μl of children's saliva using prepIT-L2P  and stored at −20°C until time of analysis. DNA was excluded from four child samples at baseline and one child and two maternal samples collected follow-up due to low quality/concentration of DNA, as measured by Nanodrop and Qubit. The level of DNA methylation was assessed  samples  rodents , 66. TheSLC6A4 gene: one cycle of 95°C for 5 min, 45 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, and 72° for 10 min. For FKBP5 gene: one cycle of 95°C for 5 min, 45 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 30 s, and 68° for 10 min.In brief, 500 ng of DNA from each sample was bisulfite converted in duplicate using the EZ DNA Methylation Gold Kit , according to manufacturer's protocol. Duplicate bisulfite-converted DNA was mixed with 0.2 μM of each primer and amplified using the HotstarTaq plus Master Mix . For each sample, PCRs were performed on each of the duplicate bisulfite treatments using the following protocol for n = 72, and follow-up children's sample n = 29–31, and mother's sample n = 30–31.DNA methylation levels for all CpG sites were assessed using the Pyromark Q24 pyrosequencer, following standard protocols . A bisulfite conversion check was included in each assay to verify full conversion of the DNA. If the difference between two bisulfite replicates exceeded two standard deviations (SDs) of the variation in the entire study population, a third bisulfite treatment was tested and the average of the two closest results was used. The final analytical sample with complete data at baseline for children ranged from 67 to 68, and for mothers Mothers self-reported at each time point their own age, marital status, number of years lived in the US, legal status, and maternal smoking status. Children reported their own age and gender at each time point.SLC6A4 and FKBP5, separately at each time point, using Pearson's correlations, to guide model building in subsequent analyses.We first tested correlations between each psychosocial stressor and resilience factor, key demographic factors, and all measures of cardiometabolic risk, and DNA methylation across all studied sites of We next modeled associations between each psychosocial stressor and resilience factor with all cardiometabolic risk markers, and separately with each methylation site, and the average across sites, in the two genes. While a formal mediation analyses was not supported by our small sample size, we also tested if the methylation levels at any site within either gene were associated with the cardiometabolic risk markers to test the hypothesis that methylation of stress-related genes may be on the pathway toward cardiometabolic disease.p-value. We tested additionally including socioeconomic and immigrant-related demographic factors, including maternal years in the US, legal status, marital status, and education level, but missing data across variables limited the value of these models, which are not presented. Where significant, we also report the effects of each of these factors given their role in contributing to the immigrant context. We chose not to adjust for multiple testing corrections, since these adjustments can be overly conservative for exploratory analyses, particularly when methylation sites are highly correlated within regions, and exposures are also correlated. All analyses were conducted in R (http://www.R-project.org).All associations were modeled using linear regressions by adjusting for a set of key covariates, including child's age, mother's age, child's gender, and maternal smoking, which are known confounders in epigenetic studies . All asst-tests. We also explored longitudinal changes in methylation with linear mixed models, adjusting for the same key demographics as other described models.In a secondary set of analyses, we examined differences in methylation levels over time in both mothers and children, as well as the overall relationship between mothers and children's methylation at each time point using paired Demographic characteristics of our analytical sample of 79 baseline and 39 follow-up CHICOS participant mothers and children are displayed in All measured stress and resilience levels at both time points are reported in Bivariate correlations between all study variables can be found in p = 0.034] (p = 0.048] and everyday discrimination  were positively associated with children's waist circumference. In the mothers at follow-up, a negative association was detected between acculturation (SASH) and BMI , and between subjective social status and SBP  , = 0.034] . At foll= 0.040] . Other mFKBP5 methylation , p = 0.008] and lower average methylation across sites of FKBP5 . Child's report of parental social support was positively associated with methylation at the first site  of FKBP5 . In mothers at baseline, various sites of SLC6A4 were positively associated with total maternal stress, IRS, health stress, and discrimination stress (p < 0.040). SSS was negatively associated with CpG1, CpG3, CpG6, and average methylation .In children at baseline, positive associations were detected between IRS with two sites in and CpG5 . ChildreSLC6A4  , while SASH was positively associated with CpG3, CpG4, CpG5, and average methylation at SLC6A4 . Mothers showed a significant decrease in FKBP5 methylation at CpG2 site  and average across sites  . For maternal methylation, no significant changes over time were detected for FKBP5 after adjusting for covariates.Children showed a significant increase over time in  0.0430) . We founSLC6A4, and greater levels of resilience factors were associated with lower methylation. While fewer associations were detected with FKBP5, generally, greater stress was associated with lower DNA methylation, and greater resilience with higher methylation. These trends were consistent with prior studies of these gene regions, where greater stress was associated with higher methylation in the promoter of SLC6A4, and lower methylation in intron 7 of FKBP5  on cardiometabolic risk and DNA methylation of stress-related genes over time. In general, stressors were associated with increased adiposity, while protective factors, such as social support and higher subjective social status were negatively associated with adiposity and BP. Associations with epigenetic factors were generally in the expected direction in both mothers and children within each gene, such that increased levels of stress were associated with higher DNA methylation in of FKBP5 , 38.Below we describe in detail the magnitude and directions of associations, how our results relate to similar studies and contribute to theories of embodiment across the life course, and the potential functional relevance of findings.We found children's stressors, such as everyday discrimination and school stress, positively associated with larger waist circumferences. This trend is consistent with other studies in adults that have shown stressors such as everyday discrimination to be associated with increased waist circumference  and low In the mothers, we found greater acculturation and resilience factors , associated with lower cardiometabolic risk, but little evidence that stress associated with either BMI or BP. A substantial literature supports associations between social support and BP and BMI; e.g., social support has been associated with lower BP reactivity to laboratory stress in older adults  and was FKBP5, a gene related to HPA axis functioning. Additionally, the serotonergic system, including methylation of SLC6A4, is typically associated with social and emotional reactivity to stress . The consistency of associations with methylation over time was unsurprising, giving the relative similar reports of stress exposures over time, and the relatively short time period of 2–3 years between samples. This consistency speaks to the relative stability of methylation data over time but also potentially the ongoing challenges faced by immigrant families over the past few decades, and the fact that more recent period of anti-immigrant focused politics in the US may not be a uniquely important stressor experienced in this population.In comparing across time points, significant epigenetic associations were found at similar frequencies at baseline and follow-up time points. In fact, in many cases the specific findings were similar across time points , our findings suggest there may be unique benefits to social support and optimism. These same factors have shown to buffer the effects of adverse childhoods on adult health behaviors and health outcomes , and thuSLC6A4 methylation was associated with more exposure variables than FKBP5. Specifically, we found very little significant associations with FKBP5 in our baseline sample. Only maternal acculturation was associated with lower methylation at this gene, in the expected direction for a stressor in FKBP5. Interestingly, we found acculturation was also associated with lower methylation in SLC6A4, though higher methylation is expected for stress in this gene. While acculturation can be a stressful process, it can also have health benefits. Our measure of acculturation was a simple measure of language use. More detailed acculturation measures in the future could help clarify what components of acculturation are acting as risk or resilience factors for health, and how they relate to these epigenetic markers.When comparing findings across tested genes, overall FKBP5, and parental social support with higher methylation. These associations were significant even after adjusting for covariates, and were in the expected direction based on prior studies where stress associates with lower methylation at this gene  at baseline, while greater subjective social stress was associated with decreased methylation in mothers. To our knowledge, SSS has never been evaluated with epigenetic data before, but appears to be protective against stress-related epigenetic changes in our findings. At follow-up, optimism was associated with lower methylation in children at this gene. In our prior research (SLC6A4) were inconsistent with baseline trends, and also inconsistent with prior studies. These findings should be interpreted with caution as they could be potentially spurious associations related to the much smaller sample size at follow-up.We found many different stressors associated with increased methylation  at research , we founSLC6A4 was significantly associated with lower BMI percentile and waist circumference in the children, but greater BMI and higher BP in the mothers, at the follow-up time point. Similar to our findings in the children, one prior study found greater methylation of a CpG site in the same region in SLC6A4 in blood of adolescents was associated with lower measures of adiposity including BMI, skin fold thickness, and waist circumference (In regards to the functional relevance of these results, one noteworthy finding was that methylation at mference . Their smference . These fSLC6A4 also associated with stress factors in our dataset, taken together with these cardiometabolic findings, our data suggest that methylation may be a pathway through which stressful experiences like discrimination can become embodied and ultimately affect cardiometabolic health. Larger and longer-term longitudinal studies will ultimately be needed to formally address the role of this gene in mediating cardiometabolic outcomes across the life course.Given that these same sites of FKBP5 and an increase in children's methylation at one CpG site in SLC6A4. For SLC6A4 specifically, we found the effect persisted after adjusting for age, suggesting the increased methylation was not just an aging effect. These longitudinal trends, though small in magnitude, are in the direction expected for increased stress in their environments. In contrast, our measures of stress did not increase over time in mothers, but they did in children, who may have become increasingly aware of adverse sociopolitical environments as they aged. While there have been increasing policies directed against Latinx people during this 2-year study period, these changes may not represent a unique moment in time for this population, who have consistently faced high levels of discrimination and anti-immigrant policies for decades. Thus, this current sociopolitical moment may be less impactful than a lifetime of exposure to these stressors experienced by the mothers, consistent with the weathering hypothesis.Over time, we found a decrease in mothers' average methylation levels at Several limitations should be noted. First, interpretation of our findings is limited by relatively small sample sizes, especially at the follow up time point. The large attrition was expected, considering how recruitment was limited by several factors regarding the socio-politically stressful time period. This is typical in studies of vulnerable populations as there are inherent difficulties in recruiting a largely undocumented immigrant population . The higOur study contains a number of unique strengths as well. First, despite a small sample size, our study was unique in the depth of collected data, which includes comprehensive measures of psychosocial stress and resilience. Our survey data were obtained through extensive 2–3-h interviews with the mothers and an hour with the children. Another strength lies in the depth of data from two perspectives—mothers and children. This is especially important as children's perspectives on stress are very underrepresented in the literature, and provide insights on different epigenetic dynamics in different life stages. Further, the longitudinal design permits comparison of stress and methylation across different sociopolitical periods and across a ~2-year span of childhood. Longitudinal methylation data are very rare, especially during a difficult sociopolitical period for this population. This study is also one of the only studies of stress and methylation in Latinos. Finally, our targeted hypothesis-driven epigenetic approach is valuable, particularly for a focused study with extensive and detailed sociocultural data that are usually very challenging to collect on larger samples necessitated by genome-wide approaches .SLC6A4 with cardiometabolic markers implies potential functional relevance of methylation at these sites in contributing to obesity and other cardiovascular or metabolic diseases. Taken all together, our findings suggest that methylation may be a pathway through which stressful experiences like discrimination can become embodied and ultimately affect cardiometabolic health.Our findings demonstrate an epigenetic pathway through which early adversity and ongoing stressful life events associate with DNA methylation within important regulatory regions of two well-characterized stress-related genes. This study explored these epigenetic associations within the context of a shifting sociopolitical environment. Many different stressors were associated with both cardiometabolic health markers and DNA methylation in both children and mothers at both genes and across time points. Associations between methylation at The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Vanderbilt University IRB-Human Research Protections Program; University of California San Diego IRB-Human Research Protections Program. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. Oral informed consent was obtained from all participants.EC and AN conceived of the study. AN coordinated and oversaw all study activities. EC generated the methylation data. All authors contributed to the analysis of the results and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Municipal incinerator bottom ash (MIBA) and sewage sludge ash (SSA) are secondary wastes produced from municipal incinerators. Landfills, disposal at sea, and agricultural use have been the major outlets for these secondary wastes. As global emphasis on sustainability arises, many have called for an increasing reuse of waste materials as valuable resources. In this study, MIBA and SSA were mixed with clay for ceramic tile manufacturing in this study. Raw materials firstly went through TCLP (Toxicity Characteristic Leaching Procedure) to ensure their feasibility for reuse. From scanning electron microscopy (SEM), clay’s smooth surface was contrasted with the porous surface of MIBA and SSA, which led to a higher water requirement for the mixing. Specimens with five MIBA mix percentages of 0%, 5%, 10%, 15%, and 20% (wt) and three SSA mix percentages of 0%, 10%, and 20% (wt) were made to compare how the two waste materials affected the quality of the final product and to what extent. Shrinkage tests showed that MIBA and SSA contributed oppositely to tile shrinkage, as more MIBA reduced tile shrinkage, while more SSA encouraged tile shrinkage. However, as the kiln temperature reached 1150 °C, the SiO2-rich SSA adversely reduced the shrinkage due to the glass phase that formed to expand the tile instead. Both MIBA and SSA increased water tile absorption and reduced its bending strength and wear resistance. Increasing the kiln temperature could effectively improve the water absorption, bending strength, and wear resistance of high MIBA and SSA mixes, as SEM showed a more compact structure at higher temperatures. However, when the temperature reached 1100 °C, more pores appeared and seemingly exhausted the benefit brought by the higher temperature. Complex interactions between kiln temperature and MIBA/SSA mix percentage bring unpredictable performance of tile shrinkage, bending strength, and water absorption, which makes it very challenging to create a sample meeting all the specification requirements. We conclude that a mix with up to 20% of SSA and 5% of MIBA could result in quality tiles meeting the requirements for interior or exterior flooring applications when the kiln temperature is carefully controlled. As the main mechanism of municipal waste treatment, incineration involving the process of combustion of wastes at high temperature  could reduce waste volume up to 90% , althougThe concrete industry, while being eager to attend to the environmental issues related to the production process such as increasing energy efficiency and the adoption of alternative raw materials, represents one of the most relevant solutions for MIBA and SSA ,7. MIBA Artificial aggregates produced by stabilization/solidification methods in a rotary plate granulator makes it possible to substitute upgraded MIBA for coarse aggregate . A maximThe concrete industry has also realized different applications of SSA, from cement component to concrete element in ground form. SSA has been successfully adopted as a fine filler aggregate . A maximIn addition to the concrete industry, the ceramic industry is another notable sector that has been reusing MIBA and SSA as valuable raw materials. In 2003, Cheeseman et al. from the UK attempted an early pioneering project and delivered a successful ceramic processing of incinerator bottom ash . ContinuAlongside MIBA, SSA has also been used for making ceramic products, such as bricks, tiles, and glass ceramics. As early as 1997, Endo et al. suggested using SSA for obtaining ceramic materials . In 20012 as an additive to tiles containing SSA. They proved that the tile bending strength improved with increased nano-SiO2 amount [In addition to using SSA alone to replace clay, some studies have started to add multiple waste materials for ceramic tile production to improve the quality of the final product. Lin et al. 2007 applied nano-SiO2 amount . Lin and2 amount . Kim et 2 amount . They fo2 amount . Esmeray2 amount .In this study, we attempted to mix municipal incinerator bottom ash (MIBA) and sewage sludge ash (SSA) with clay for making ceramic tiles, and we investigate how the two materials affect the quality of the final product and to what extent. Specimens with different mixes of replacement levels of MIBA, SSA, and clay were prepared and calcined at different temperatures. By conducting a series of macroscopic tests, the research team made an effort to determine appropriate combinations of raw materials at suitable sintering temperatures so that final products with a satisfactory quality level for different applications could be delivered.This section reports the basic properties of the three raw materials applied to this study. SEM (scanning electron microscopy) is used to show the microstructure of the materials. SEM analysis was conducted in the Micro and Nanostructure Analysis lab at I-Shou University, and the SEM equipment used was HITACHI-S4700 . The Hitachi S-4700 is a field emission scanning electron microscope (FE-SEM), which is more powerful than a standard SEM. The S-4700, under optimal conditions, can magnify images upwards of 500,000 times and resolve features to 2 nanometers. The signals generated during SEM analysis produce a two-dimensional image and reveal its internal morphology (texture). 2, CaO, and Al2O3, which can also be found in MIBA and SSA [2), calcite (CaCO3), gehlenite (Ca2Al2SiO7), and hematite (Fe2O3) [3. Oxides commonly present in ceramic materials including SiO and SSA ,35,36,37 and SSA ,39,40 an and SSA ,42,43,44 (Fe2O3) ,18,19. ITCLP (Toxicity Characteristic Leaching Procedure) is a chemical analysis process to determine whether hazardous elements are present in a waste material. To recycle the MIBA and SSA as acceptable construction material, their TCLP test should yield results meeting the hazardous industrial waste standards. For this purpose, we followed the Taiwan EPA TCLP method-NIEA R201.15C, which corresponds to USEPA method 1311. The largest amount of heavy metal found in MIBA was Cu (1.57 mg/L), followed by Ba (0.658 mg/L) and Cd (<0.1 mg/L). Moreover, the largest amount of heavy metal in SSA was also Cu (4 mg/L), followed also by Ba (<0.2 mg/L). No other harmful heavy metals were detectable in MIBA and SSA, suggesting that our MIBA and SSA were suitable for recycling.3 and compressed at a 34.32 ± 0.5 MPa pressure to produce the final specimens. Thirty tile samples were contained in each set of specimens, and they were ready for use in the following tests. Specimens with five MIBA mix percentages of 0%, 5%, 10%, 15%, and 20% (wt) and three SSA mix percentages of 0%, 10%, and 20% (wt) were made so that a total of 15 sets of specimens were prepared. Prior to making specimens, all three raw materials went through an Atterberg test and found their plasticity limits in order to determine a proper mixing water amount. During the mixing process, proper compositions of clay, MIBA, and SSA were carefully prepared and kneaded in a shaft clay mixer, and a de-airing vacuum pug mill was used to expel internal pores. Finally, the properly kneaded mixtures were placed in a mold of 12 ∗ 6 ∗ 1 cmTo study how various MIBA and SSA replacements affect the amount of water applied, we used the Atterberg limits test to obtain plasticity limits of the different mixes according to CNS 5088 . Figure 2 was the main composition for SSA, and the quartz phase and glass phase were the main structures for SiO2. When the glass phase occupied more in SiO2, the surface of the tile specimens became glassy, and the air would be trapped inside the glassy surface, producing more pores in the tile specimens during the calcined process. Hence, the tile body became expanded easily. In this study, the SSA may contain more glass phase in SiO2. When the kiln temperature reached 1150 °C, the tile body started to melt and a glass foam in the body was expanded, which is caused by part of the glass phase in SiO2, as shown in Shrinkage tests were done according to CNS3299-2 . Figure 2 in SSA seemed to be building a stronger structure, which improves the melting temperature of tile specimens and becomes harder to burn out. Specific gravity was measured according to CNS 3299-3 . Figure Water absorption tests were conducted according to CNS3299-3 . Figure 2 of SSA produced large pores by air trapped in the tile specimens, resulting in a dramatic bending strength reduction. Bending strength tests were conducted according to CNS3299-4 . Figure 2 in SSA improved the hardness of floor tile specimens, and the amount of wear decreased with increasing amounts of SSA. At 1100 °C, the amount of wear increased first and then reduced with the increasing amount of SSA replacement. When the kiln temperature reached 1150 °C, melting of the tile specimens was observed, and foam was produced in the tile specimens. The amount of tile wear increased at this kiln temperature. Furthermore, the glass phase produced by SiO2 in SSA could trap air inside tile specimens and affect the compactness of the tile body. The amount of tile wear increased with the increasing amount of SSA at 1150 °C.Wear resistance tests were conducted according to ASTM D968. From 2 was formed, and a shiny surface of tile specimens was observed. 2. Since the dominant component of SSA was also SiO2, the amount of SiO2 in tiles increased with the increasing amount of SSA added. Compared with results of bending strength tests, when the amount of Ca(Al2Si2O8) produced from the combination of SiO2, CaO, and Al2O3 increased, the bending strength of tile specimens reduced. When same kiln temperature was considered, it could come to the conclusion that the bending strength decreased with more MIBA added. 29Si Nuclear Magnetic Resonance) is used to examine structural properties of porous silicon compounds in the tile specimens. The superscript n of Qn refers to the number of connections of a silicon atom to other silicon species. The Q0 type refers to a silicon atom with no connections to another silicon species, the Q1 type refers to a silicon atom connected to one silicon O-Si species, and so forth. 4. The peak value of Q4 reduced with more SSA. The values of Q4 after integration increased with more SSA added. It suggests that the addition of SSA helped improve the development of SiO4 tetrahedral structure. Si-NMR (4 became apparent with increasing kiln temperature. It suggests that the increase of kiln temperature helped Si atoms improve the development of silicate structures. 4 after integration increased first and then reduced with increasing kiln temperature. The highest value of Q4 was obtained at 1100 °C. It suggests that the kiln temperature of 1100 °C helped improve the development of SiO4 tetrahedral structure. This result conformed with those obtained from bending strength tests.The results obtained from Si-NMR analysis for floor tiles that contained 20% SSA and 20% MIBA replacements at various kiln temperatures also show that the peak values for Q2, which resulted in a production of glass phase on the surface of specimens that trapped air and produced pores at the interior of the tile body. In this study, the water absorption for all floor tile specimens met the standard of type III, and the qualifying rate increased with increasing kiln temperature. However, the qualifying rate reduced with increasing amounts of MIBA and SSA mixes. According to CNS 9737 R1018 , bending2, the use of SSA could increase the shrinkage rate of floor tile specimens within a kiln temperature of 1000–1050 °C. However, when kiln temperature reached 1150 °C, the glass phase surface formed, the tile bodies were expanded, and the shrinkage rate was reversed.The application of MIBA could reduce the shrinkage rate of floor tile specimens. On the other hand, due to the fact that the SSA contained SiO2 in SSA could improve the melting temperature of tile specimens and was hard to be burnt out; the weight loss on ignition of tile specimens increased with more SSA.Since a large amount of organic, non-organic matters, and heavy metals in MIBA were easily burned at high kiln temperatures, the weight loss on ignition of floor tile specimens increased with more MIBA. In contrast, a large amount of SiOIn general, the water absorption of tile specimens increased with increasing amounts of MIBA and SSA, but the water absorption reduced with the increasing kiln temperature. When the kiln temperature reached 1150 °C, the tile surface became vitreous and shiny, and it prevented water penetration, so that the water absorption was reduced to close zero.The bending strength of floor tile specimens reduced with more MIBA and SSA replacements. Moreover, the tile bending strength increased with increasing kiln temperature within the range of 1000–1100 °C and would achieve the maximum strength at 1100 °C. However, when the kiln temperature reached 1150 °C, foam was produced in the tile specimens and so forth, resulting in the reduction of bending strength.Tile wear increased with more MIBA replacement, while the tile wear decreased with more SSA replacement at kiln temperatures of 1000 and 1050 °C. The amount of wear increased first and then reduced with more SSA replacement at 1100 °C. However, when the kiln temperature reached 1150 °C, the amount of tile wear increased. The amount of tile wear for tile specimens increased with more SSA and MIBA replacements at 1150 °C.When the kiln temperature was considered as the main parameter, in general, the compactness of the tile body was improved by the increasing kiln temperature. At a kiln temperature of 1100 °C, the amount of pores increased with more MIBA and SSA replacements, and more pores were observed in the tile bodies from SEM images.4 became apparent with increasing kiln temperature. It suggests that the increase of kiln temperature helped Si atoms improve the development of the silicate structure. The values of Q4 after integration increased first and then reduced with increasing kiln temperature. The highest value of Q4 was obtained at 1100 °C. It suggests that the kiln temperature of 1100 °C helped improve the development of a SiO4 tetrahedral structure. This result conformed with that obtained from bending strength tests.The results obtained from Si-NMR analysis show that the peak values for QAs SSA and MIBA have been proven to be feasible replacement materials for ceramic production, the study makes a pioneering attempt to mix both materials. The interactions between SSA and MISA at various kiln temperature are complicated, but this study has come up with some encouraging results, and numerous feasible applications are suggested in


Hippocampal plasticity is hypothesized to play a role in the etiopathogenesis of depression and the antidepressant effect of medications. One form of plasticity that is unique to the hippocampus and is involved in depression-related behaviors in animal models is adult neurogenesis. While chronic electroconvulsive shock (ECS) strongly promotes neurogenesis, less is known about its acute effects and little is known about the neurogenic effects of other forms of stimulation therapy, such as repetitive transcranial magnetic stimulation (rTMS). Here, we investigated the time course of acute ECS and rTMS effects on markers of cell proliferation and neurogenesis in the adult hippocampus. Mice were subjected to a single session of ECS, 10 Hz rTMS (10–rTMS), or intermittent theta burst stimulation (iTBS). Mice in both TMS groups were injected with BrdU 2 days before stimulation to label immature cells. One, 3, or 7 days later, hippocampi were collected and immunostained for BrdU + cells, actively proliferating PCNA + cells, and immature DCX + neurons. Following ECS, mice displayed a transient increase in cell proliferation at 3 days post-stimulation. At 7 days post–stimulation there was an elevation in the number of proliferating neuronal precursor cells (PCNA + DCX +), specifically in the ventral hippocampus. iTBS and rTMS did not alter the number of BrdU + cells, proliferating cells, or immature neurons at any of the post-stimulation time points. Our results suggest that neurostimulation treatments exert different effects on hippocampal neurogenesis, where ECS may have greater neurogenic potential than iTBS and 10–rTMS. Adult hippocampal neurogenesis provides a source of cellular and synaptic plasticity that may be relevant for treating a number of psychiatric disorders . A consiNeurostimulation therapies have been used clinically for many years and continue to evolve at a rapid pace. Moreover, there are varying degrees of evidence that different neurostimulation therapies modulate hippocampal function, including neurogenesis. For example, electroconvulsive therapy (ECT) is a non-invasive treatment that triggers a controlled tonic–clonic generalized seizure using a brief electrical current under general anesthesia. It is the most efficacious treatment for severe depression and is also indicated for schizophrenia, catatonia, and active suicidal ideation ,13,14,15In contrast, transcranial magnetic stimulation (TMS) is a newer form of neurostimulation that depolarizes neurons via a changing magnetic field. TMS does not induce seizures or require anesthesia, is better tolerated than ECT  and is tAnother form of rTMS, intermittent theta-burst stimulation (iTBS), delivers bursts of stimuli according to a theta pattern (~5 Hz) ,30. iTBSNeurogenesis is a multistep process involving the proliferation of neural precursors and the survival of post-mitotic neurons through multiple distinct critical periods. Identifying the net neurogenic effect of any manipulation therefore requires an assessment of its impact on each of these stages of neuron production. A number of studies have found that ECS dramatically increases cell proliferation ,16,17 anTo address these gaps, here, we systematically examined how acute ECS, rTMS, and iTBS affect adult neurogenesis in the dorsal and ventral hippocampus. We focused on cell proliferation and the population of immature neurons since these developmental stages have been virtually uncharacterized with respect to acute ECS and have never been investigated in rTMS or iTBS models. For rTMS and iTBS, we also examined BrdU-labeled cells that were largely born shortly before stimulation. Our results provide the first side-by-side comparison of the neurogenic efficacy of these neurostimulation methods and indicate that, whereas ECS transiently promoted cell proliferation, iTBS and rTMS did not significantly alter any measures of adult neurogenesis.n = 2–4/timepoint) and we did not expect or observe any changes in numbers of proliferating cells or immature neurons across days in the absence of stimulation, we pooled all the sham animals from the various timepoints into a single group for comparison with the ECS groups.All procedures were approved by the Animal Care Committee at the University of British Columbia and conducted in accordance with the Canadian Council on Animal Care guidelines. Wild-type C57BL/6 mice were bred inhouse at the Centre for Disease Modeling at the University of British Columbia. Both male and female mice were used; ECS experiments included 21 males and 26 females; TMS experiments included 22 males and 24 females. All mice were 10 to 12 weeks of age at the time of stimulation. Mice had ad libitum access to food and water and were housed on a 12 h light/dark cycle with lights on at 7 am. Animals were handled during the week before stimulation and either underwent three days of ear clip habituation  or one week of restraint and coil placement . Mice were given one stimulation session and brains were collected for histological analyses of neurogenesis at 1, 3, or 7 days following stimulation A. To birEleven to 15-week old mice were arbitrarily assigned to ECS and sham groups, with the exception that male and female mice were equally distributed across groups. Bilateral auricular ECS was administered via padded ear clip electrodes connected to a pulse generator . Ear clip electrodes were soaked in saline solution and placed on the base of the pinna. Stimulation parameters were adapted from Yanpellewar et al.  and set Mice were 11 to 12 weeks of age at the time of stimulation. Animals were restrained to prevent movement and were stimulated with a MagPro X100 device and a TMS coil adapted for rodents . The average motor threshold was determined with a separate group of animals, before the main experiment, by stimulation with increasing power until a hindlimb motor response was evoked. Experimental stimulation power was then defined as 115% of the motor threshold.rTMS was delivered at 10 Hz, with a 5 sec train duration and a 15 s intertrain interval over 12 trains, for a total of 600 pulses B. The toMice were transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline . Brains remained in paraformaldehyde for 48 h before sectioning. A vibratome  was used to section brains into 40 µm thick coronal sections. Brain sections were stored in cryoprotectant at −20 °C until immunohistochemical processing. BrdU was used to investigate effects of rTMS/iTBS on the survival of cells born prior to stimulation, as in . Prolife3) were then obtained by dividing cell counts by the tissue volume that was sampled.Positively labeled cells in the granule cell layer and the sub granular zone were manually counted on a light microscope with a 40x objective. Four hippocampal sections from each animal were chosen , and all positive cells were counted. Certain tissue was exhausted during the course of the experiment, and thus not all animals were examined for all neurogenesis markers. Tissue volume was obtained by tracing the 2–dimensional area of the granule cell layer and multiplying by the tissue thickness, and cell densities  in size at a z–resolution of 1.5 µm were merged to encompass the entire dentate gyrus. Dorsal and ventral dentate gyrus sections were counted in Image J (with the Cell Counter plugin) for cells positive for PCNA and cells positive for both PCNA and DCX. The percentage of neuronal precursors was defined as the percentage of PCNA positive cells that were also DCX positive. The average percentage of neuronal precursors of each stimulation group was obtained and used to extrapolate the density of neuronal precursors.Statistical analysis was performed using Prism 8 , R, and SPSS. Cell counts in the ECS and rTMS/iTBS experiments were analyzed by two-way  repeated measures ANOVA. In cases where tissue was not available for both subregions of a given animal, data were analyzed by mixed effects analysis. Where animal ages were not evenly distributed across groups, data were analyzed by ANCOVA, with age as a covariate. Where significant effects were observed, group differences were examined further by post hoc Tukey HSD tests. The statistical significance threshold was set at α = 0.05.3,37 = 3.1, p = 0.037; dorsoventral effect: F1,37 = 2.6, p = 0.11; dorsoventral interactions all p > 0.8; sham vs. day 3: p = 0.015, all other comparisons p > 0.17; To determine whether acute ECS elevates cell proliferation at specific timepoints, we quantified PCNA+ cells at 1, 3, and 7 days post-stimulation and analyzed by repeated measures ANCOVA , with age as a covariate since animal ages were not evenly distributed across experimental groups. PCNA+ cell densities progressively increased from days 1 to 3 post-ECS and were ~60% greater than shams at day 3. By day 7, the density of PCNA+ cells declined and was comparable to sham levels. PCNA cell densities did not differ along the dorsoventral axis . Post hoc analyses revealed greater numbers of double positive PCNA + DCX + cells on day 7 compared to shams (sham vs ECS days 1/3/7: p = 0.2/0.8/0.004), an effect that was specific to the ventral DG .The PCNA+ cell population is heterogeneous and composed of multiple types of dividing cells. To determine whether ECS specifically alters the proliferation of neuronal precursors, we quantified the proportion of PCNA + cells that also expressed the immature neuronal marker DCX. Here, we found that 20–40% of PCNA + cells also expressed DCX, with more cells expressing DCX on days 1 and 7 compared to day 3 , exploratory analyses suggest that the decline in BrdU + cells over days appeared to be driven by the iTBS group, which had more than twice as many BrdU + cells on day 1 compared to day 7 .Stimulated mice were examined at 1, 3, or 7 days post-stimulation and compared to sham-stimulated mice. Stimulation did not differentially modulate markers of neurogenesis across the dorsoventral axis  and thus2,39 = 1.4, p = 0.3; day × group interaction: F4,39 = 0.9, p = 0.5; PCNA + cell densities were analyzed to determine rTMS and iTBS–related changes in cell proliferation. Here, no differences were observed across groups . Here, we found that both iTBS and rTMS did not alter BrdU + cells, though our exploratory analyses suggest that recently born neurons may be culled at a faster rate following iTBS. Collectively, these findings provide new insights into the temporal specificity and efficacy by which different neurostimulation modalities impact neurogenesis.ECS is arguably one of the most robust neurogenic stimuli identified to date and typically elevates neurogenesis by 2–3-fold compared to baseline levels ,45,46. NHere, we used a single stimulus to refine the exact point at which ECS promotes proliferation in mice. Previous studies in rats have reported that a single ECS treatment can promote proliferation 2–3 days later ,41,46,51In contrast to its effects on proliferation, ECS did not significantly alter the density of immature DCX + cells during the week, following stimulation. This may be because the transient rise in proliferation produced a relatively small increase in new cells compared to the large number of immature neurons that express DCX. Alternatively, we did observe an increase in the density of proliferating neuronal precursors at 7 days post-ECS and thus it is possible that there is a delayed increase in neuron production due to the time required for seizures to promote the division of radial stem cells and birth of the transit amplifying cells that are ultimately responsible for producing newborn neurons . Future Whereas ECS directly depolarizes large populations of neurons through applied current and leads to generalized seizures, TMS induces milder degree of neuronal depolarization via a changing magnetic field and does not trigger seizures . While tBy all measures, TMS had weaker effects on neurogenesis measures than ECS. Whereas chronic rTMS has been found to increase neurogenesis in several studies ,27, a siOur experimental design intended to maximize similitudes with clinical applications of neurostimulation treatments in humans in an effort to increase the translational interpretation of our results . Interest in neurogenic treatments stems from findings that hippocampal dysfunction, including structural atrophy and volume loss, is observed in multiple disorders such as depression , schizopWhile there are debates regarding the magnitude of adult hippocampal neurogenesis ,79,80,81There are several limitations of this study that are important to consider. First, compared to clinical settings, neurostimulation treatments have low spatial resolution in mouse models. Whereas TMS typically targets the dorsolateral prefrontal cortex in humans, modeling work in rodents estimates that our TMS coil generates a magnetic field that likely directly stimulates the entire mouse brain , includiSecond, due to tissue limitations, we relied on cell density measurements rather than stereological cell quantification. This may be an issue if treatments induced changes in tissue volume. However, ECT increases hippocampal volume in patients , which wFinally, we injected BrdU 2 days before rTMS and iTBS to monitor survival of immature cells. While this will primarily label cells born before stimulation, it is possible that a small number of label-retaining precursor cells may have been dividing at the time of stimulation , explainIn summary, among the three neurostimulation methods tested, neurogenic effects are robust following acute ECS, and weak or absent following acute iTBS and rTMS. Given that rTMS can promote neurogenesis in chronic settings, it will be worthwhile to investigate whether chronic iTBS also influences neurogenesis to a similar extent. Given the high degree of plasticity of newborn neurons, it will also be important for future studies to examine the functional properties of neurons that are subjected to neurostimulation and determine their role in the memory- and mood-related functions of the hippocampus.