Inflammatory bowel disease (IBD) refers to chronic intestinal immune-mediated diseases including two main disease manifestations: ulcerative colitis (UC) and Crohn’s disease (CD). Epidemiological, clinical, and preclinical evidence has highlighted the potential anti-inflammatory properties of naturally occurring alkaloids. In the present study, we investigated the potential anti-inflammatory activities of the tobacco alkaloids nicotine and anatabine in a dextran sulfate sodium (DSS)-induced UC mouse model with a fully humanized immune system. Our results show that nicotine significantly reduced all acute colitis symptoms and improved colitis-specific endpoints, including histopathologically assessed colon inflammation, tissue damage, and mononuclear cell infiltration. The tobacco alkaloid anatabine showed similar effectiveness trends, although they were generally weaker or not significant. Gene expression analysis in the context of biological network models of IBD further pinpointed a possible mechanism by which nicotine attenuated DSS-induced colitis in humanized mice. The current study enables further investigation of possible molecular mechanisms by which tobacco alkaloids attenuate UC symptoms. Inflammatory bowel disease (IBD) is the collective term for chronic idiopathic immune-mediated diseases characterized by chronic and relapsing inflammation of the gastrointestinal tract. The incidence and prevalence of IBD have increased worldwide with a stable rise expected over the next decade, which will substantially increase the burden on health care systems and society ,2. IBD cThe correlation between cigarette smoking and IBD has been established by several epidemiological studies . While sSeveral preclinical and clinical studies have been published in which nicotine was administered to mice or patients with UC in different formulations using various administration routes ,18,19,20In the present study, to increase the human translatability of a colitis mouse model and investigate the potential role of tobacco alkaloids in reducing intestinal inflammation, we combined a mouse model with a fully humanized immune system and a validated DSS-induced mouse model of UC with disease-specific endpoints and system-wide molecular profiling.To mimic the contributions of the human immune system in a preclinical model, we engrafted immunodeficient mice (NCG) with cord-blood-derived human CD34+ hematopoietic stem cells and progenitor cells. Fourteen weeks after stem cell injection, engraftment level was monitored with flow cytometry analysis of human CD45+ cells among total blood leukocytes (mouse and human). Only mice with a humanization rate > 30% were used in the subsequent experiments (hu-mice). The average humanization rate was 52.9%. The engrafted human stem cells matured and developed a fully functional human immune system containing mostly B and T lymphocytes, but also monocytes, natural killer (NK) cells, and neutrophils A; T lympThe mice were treated with nicotine or anatabine for 2 weeks before colitis induction by DSS until the end of the experimental phases at day 7 (acute phase) or 12 (7 days DSS + 5 days replacing DSS with water ad libitum—recovery phase).Treatment with alkaloids did not induce significant body weight loss before DSS induction. Overall survival was recorded during the 12-day experiment. Mice treated with nicotine did not show DSS-induced mortality, but five mice in the vehicle (drinking water) and three mice in the anatabine arms died by the end of the experimental recovery phase (day 12) .After colitis induction, body weight started to decrease and reached a loss of 22% in the vehicle-treated group. Anatabine and nicotine attenuated body weight loss, albeit not significantly . DiarrheThe three regions  in the Swiss-roll colon samples A were quFlow cytometry was performed to determine the effects of nicotine and anatabine on human immune cell populations in the blood samples collected at days 7 and 12. The number of circulating human immune cells (hCD45+) was not significantly affected by treatments on day 7 or on day 12 A. The nuTo assess human immune cell infiltration in the colon of DSS-treated hu-mice, the fixed colons were stained to label T cells, B cells, macrophages, neutrophils, and proliferative cells . AnalysiAs the samples used for RNA sequencing contained material from both mouse and human origins, we set up a dedicated preprocessing pipeline for the raw sequencing data see . Supplemp-value and directionality of the inferred regulation by nicotine at day 6 in To obtain mechanistic insight into nicotine’s effects on DSS-induced colitis, we used an enrichment approach that relies on a database of over 800 molecular entities or nodes  with mRNAs known to be regulated by the nodes. The activity of these nodes can be inferred by comparing differential mRNA regulation in experimental data to the mRNAs assigned under each node, taking into consideration the expected directionality of the regulation ,35,36. HFirst, we investigated the pathways regulating nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB) complex—inferred to be downregulated—in the merged IBD network model A. Along Next, we investigated the impact of other pathways inferred to be regulated by nicotine in the DSS background and leading to intestinal permeability, wound healing, and inflammation. The inferred upregulation of Janus kinase (Jak) and downregulation of mitogen-activated protein kinase (Map3k)5 led to reduced intestinal permeability in nicotine-treated mice via increased translocation of beta catenin (Ctnnb)1 from intracellular space to tight junctions and decreased translocation of occludin (Ocln) and the tight junction protein (Tjp)1 from tight junctions to extracellular space, respectively B. IntereTraf3ip2 (Traf3 interacting protein 2) was inferred to be downregulated, suggesting increased cell proliferation and wound healing B. Enhanc−/− mice transplanted with human cord-blood-derived CD34+ hematopoietic stem cells develop a naïve and functional human immune system equipped with T and B cells, NK cells, dendritic cells, monocytes, and macrophages. Finally, our study design also considered the relapsing and remitting characteristics of human CD and UC with the addition of a 5-day recovery period following the 7-day DSS treatment period (referred to as the recovery phase). This is important to evaluate the alkaloids’ effects on both the pro-inflammatory (acute) and anti-inflammatory/pro-wound healing (recovery) phases of the disease [Naturally occurring alkaloids in tobacco have been suggested to be potentially effective in treating several inflammatory conditions ,27,39,40 disease ,46.In this setting, our results clearly show that oral administration of nicotine and partially anatabine globally reduced the clinical manifestations of DSS-induced colitis accompanied by body weight loss, diarrhea, rectal bleeding, and colon shortening. Furthermore, we were able to link observed changes in the clinical outcomes to direct effects at the tissue level. The histopathological assessments confirmed the role of nicotine in reducing the inflammation, tissue damage, and mononuclear infiltration scores ,23,47 duThe node inference and network analysis pinpointed mechanisms known to be regulated by nicotine. In line with our findings, several studies have shown that the suppressive effects of nicotine on the production of inflammatory mediators such as Tnf and Il1b occur via inhibition of the NF-ĸB pathway ,50,51,52The inferred downregulation of Tlr2 (upstream of the NF-ĸB complex) could result from nAchR activation, which has been shown to attenuate Tlr signaling. Topically administered nicotine was shown to decrease Tlr2 production in diabetic wounds infected with bacterial agents and reduce the inflammatory response during skin wound healing ,58.Our analysis also predicted nicotine-mediated downregulation of Tlr7 in DSS-treated mouse colon. According to the network model, Tlr7 downregulation leads to downregulation of Ifnb1, which inhibits anti-inflammatory mechanisms. Although this is initially counterintuitive, the evidence shows opposite effects elicited by TLR7 activation/inhibition in the context of inflammation. Although TLR7 activation has been shown to protect mice from DSS colitis by mediating the action of enteric viruses ,60, its MAPKp38 is connected to epithelial cell migration in our IBD network models, and its inferred downregulation would indicate impaired wound healing. However, some studies have suggested that MAPKp38 is activated in the inflamed intestinal mucosa , and itsIn the IBD network model, vitamin D signaling downregulates the NF-κB component, Rela , and itsThe study has its limitations. Due to the lack of an exposure arm without DSS treatment in the study design, it was not possible to investigate how tobacco alkaloids may impact the healthy colon in the humanized mouse model. Moreover, analysis of the human-cell-derived transcriptome was not possible due to the small sample sizes extracted from the colon of the alkaloid-treated DSS mice. Even though the changes in immune cell infiltration to the colon at day 7 were marginal in the tobacco-alkaloid- and DSS-treated groups compared to mice treated with DSS only, the immune cells likely signal to the colon epithelium. Hence, the analysis of the immune cell transcriptome in the humanized mouse model could further explain the inferred molecular changes observed in the colon transcriptomic analysis and increase understanding of how tobacco alkaloids protect the mouse colon. Nevertheless, our results can be used to generate limited hypotheses on the mode of action of nicotine in experimental colitis, which should be confirmed with additional investigations.In summary, we demonstrated that administration of nicotine in drinking water reduced colitis clinical symptoms and histologic features in a DSS-mouse model with a fully humanized immune system. The underlying molecular mechanisms were linked to known effects of nAchRα7 signaling, and the node inference also suggested novel mechanisms of nicotine action that could be further explored.All animal procedures described in this study were reviewed and approved by the local ethic committee . The experiment was carried out with NOD/Shi-scid/IL-2Rγnull immunodeficient mouse strain . Briefly, 4-week-old immunodeficient NCG mice were engrafted with cord-blood-derived human CD34+ hematopoietic stem and progenitor cells  2 days after chemical myeloablative treatment. Fourteen weeks after cell injection, engraftment level was monitored with the analysis of human CD45+ cells among total blood leukocytes by flow cytometry . After engraftment confirmation, mice were assigned to each treatment group after manual randomized based on their humanization rate, their weight, and the cord blood donor ID.https://doi.org/10.26126/intervals.7p6n5w.1.Acute colitis was induced by adding DSS  in drinking water over 7 days, from day 1 to day 7. DSS powder was aliquoted on the first day of the experiment. DSS solution was freshly prepared by dissolving the aliquoted powder in MilliQ water , and it was renewed every 2 days. For the washout period, the DSS solution was removed and replaced by MilliQ water from day 8 to day 12. The progression of the disease severity was monitored with clinical scoring. Mice were weighed daily during the acute colitis cycle and scored for their stool consistency and rectal bleeding by visually monitoring stool. Using these parameters, a global clinical score was calculated  and anatabine  with a purity of 99%) were added to the drinking water to reach a value of 20 mg/kg/day.Flow cytometry analysis was performed on an Attune N × T Flow Cytometer (Life Technologies/Thermo Fisher Scientific). Briefly, 100 µL of blood was stained with surface antibodies  and viability dye. Red blood cells were lysed with RBC lysis buffer . Samples were then permeabilized with Fixation/Permeabilization Buffer (Thermo Fisher Scientific) for the intracellular staining. Washing steps preceded cytometer analysis. The numbers of samples were n = 10 at D7 for all groups, n = 7 for the water and anatabine groups at D12, and n = 10 for the nicotine group at D12.Distal colon tissues were collected in Magnalyser tubes  and ground in 700 µL of Qiazol lysis buffer  with the FastPrep-24 5G . Total RNAs (including microRNAs) were purified using miRNeasy Mini Kit  according to the manufacturer’s instructions.RNA concentration and purity were determined using a UV spectrophotometer  by measuring the absorbance at 230, 260, and 280 nm. The integrity of the RNA was further checked with the Agilent 2100 Bioanalyzer, and RNAs with RIN values > 7 were used in the downstream RNAseq workflow.The RNA was normalized to 200 ng for each sample, and the library was prepared with the Tecan Universal Plus mRNA-Seq Kit . Briefly, this kit generates stranded RNA-Seq libraries derived from poly(A)-selected RNA. The PolyA RNA was reverse-transcribed and transformed into a double-stranded cDNA. Unique dual-indexed adapters were ligated, and libraries were then amplified with 10 polymerase chain reaction cycles and purified. The quality of the libraries was also checked on a Fragment Analyzer 96 instrument . The purified libraries were then quantified with NuQuant and normalized to 10 nM. Eight sequencing pools were clustered on a c-Bot instrument and sequenced on the Illumina HiSeq 4000 Instrument .Colons were dissected, measured, and fixed in 4% paraformaldehyde for 24 h before storage in 70% ethanol. Samples were dehydrated before paraffin embedding and trimming using a Leica TP1020 Tissue Processor . Each colon sample was embedded in one paraffin block to perform longitudinal sections. Sections of 4 μm per block were deposited on Superfrost + slides before HE staining. Two sections of the same block were used to prevent artifacts from manual coloring. Analysis was performed on mice colon tissues prepared with the Swiss-roll technique. All slides were digitalized using the scanner Aperio Versa (Leica) with the ×20 objective in brightfield conditions. Inflammation/fibrosis, tissue damage, and mononuclear cell infiltration were then visually assessed in proximal, middle, and distal regions and scored . One-way ANOVA with Dunnett’s multiple comparison test was used, and the results are shown in The raw sequencing data were first cleaned to remove low-quality and adapter sequences. Downstream analysis revealed that mouse RNA was much more abundant than human RNA in the collected samples . The validity of the mapping strategy was assessed by running an over-representation analysis of the top 1000 genes contained in each tail of the distribution of the log10 ratio between the mean read counts in human and mouse for orthologous genes. The tissue-specific gene set collections “Human_Gene_Atlas” and “Mouse_Gene_Atlas” were taken from the EnrichR website . The gene set over-representation calculation was performed using the piano package [Mapped transcriptomic data were processed in the R software environment for statistical computing . The hum package . Once thp-value < 0.05 were then mapped to the IBD network model suit using Cytoscape [Node enrichment analysis was used to identify molecular drivers of nicotine effects on DSS-induced colitis. The strength scoring algorithm is a threshold-free enrichment method that relies on mRNAs known to be regulated by specific nodes, as described in ,76. The ytoscape . The graytoscape . The IBDytoscape ,38.


Lambl's excrescences (LEs) are delicate filiform strands formed by connective tissue located along the valve closure lines within the cardiovascular system. Most cases of these excrescences manifest without discernible symptoms, and the exact etiological factors contributing to their formation remain unknown. These excrescences may embolize to the brain, causing strokes. It is essential that all other possible causes of stroke be eliminated prior to identifying Lambl's excrescences as the cause of the stroke. Herein, we present a case of a patient who suffered a stroke, and all conventional testing for common causes of embolic strokes was ruled out. In pursuit of a comprehensive evaluation encompassing the classification of the stroke as cryptogenic, a transesophageal echocardiogram (TEE) was performed, which effectively disclosed the existence of LEs situated on the aortic valve leaflets. The patient was treated with anticoagulation and discharged with close follow-up monitoring. To culminate, the inclusion of this case within our study augments the currently scarce pool of instances that exhibit such characteristic cardioembolic phenomena, thereby accentuating the necessity for additional prospective investigations to substantiate the existence of a causative association linking LEs and cardioembolic strokes. Stroke is the leading cause of long-term disability and the second leading cause of death worldwide, resulting in the death of 26 million people each year . A strokLambl's excrescences were first described as a cause of embolic stroke in 1856 by Vilém Dušan Lambl, a Bohemian physician . In the A 53-year-old female presented to the emergency department (ED) with weakness in her left upper and lower extremities, a drooping left side of her face, and difficulty speaking. She did not have a significant medical history or a surgical history, nor had she traveled recently. According to the patient, she has never smoked, consumed alcohol, or used illicit drugs. The physical examination was unremarkable except for the neurological examination, which showed dysarthria, facial asymmetry, and weakness in the left lower and upper extremities. The National Institutes of Health Stroke Scale (NIHSS) score was 11 . The patient was out of the tissue plasminogen activator (tPA) window.The patient's laboratory results were not significant at the time of admission. The urine toxicology screen was negative, and the lipid panel was within the reference range. Electrocardiography (EKG) showed sinus bradycardia with a normal rhythm Figure .CT scan of the head without contrast was performed, which indicated no acute intracranial pathology. A CT angiogram with contrast of the head and neck revealed occlusion of the supraclinoid internal carotid artery on the right, as well as cross-filling of the right A2 segment and the distal M1 segment of the middle cerebral artery (MCA) as shown in Figure The patient was immediately taken for percutaneous transluminal mechanical thrombectomy of the right MCA, followed by treatment with aspirin, clopidogrel, and deep vein thrombosis (DVT) prophylaxis with heparin. After the procedure, a CT scan of the head without contrast revealed an acute infarct in the right frontal lobe and right basal ganglia. Additionally, a mass effect was observed on the right lateral ventricle without a shift in the midline. Following the procedure, the patient's left lower extremity weakness and left upper extremity weakness significantly improved; however, facial droop remains.A radiological and hematological examination was conducted to identify the source of the embolus. A transthoracic echocardiogram (TTE) demonstrated a normal left ventricle size and left ventricle thickness above the normal range. During early diastole, the left ventricle displayed decreased relaxation. The TTE revealed no evidence of clots in the heart. A brain MRI without contrast was performed, which showed acute stroke in multifocal areas within the right cerebral hemisphere primarily in the inferior frontal lobe Figure .A bubble study was performed and revealed a small patent foramen ovale. There were negative results for bilateral Doppler ultrasounds of the lower extremities, bilateral renal ultrasounds, and a Holter study of the lower extremities. All coagulation tests, including factor V Leiden, lupus anticoagulant, anticardiolipin, antithrombin III activity, protein C and S resistance, prothrombin G20210A mutation, anti-nuclear antibody (ANA), and homocysteine levels, were negative. A transesophageal echocardiogram (TEE) was performed which revealed a fine filamentous strand (6 mm in length and 1 mm in thickness) on the aortic valve consistent with Lambl's excrescence Figure , a rare The patient was hospitalized for 14 days, and the remainder of his stay was uneventful. The patient was discharged on a regimen of aspirin, ticagrelor, and atorvastatin. The patient was discharged to a rehabilitation facility for acute rehabilitation.The first description of Lambl's excrescences (LEs) came from Bohemian physician Vilém Dušan Lambl. Initially, these lesions were identified on the aortic valve as filiform lesions . While LThe aortic valve and mitral valve represent the two most common locations for LE, with the aortic valve being the chief source of stroke in LE . In a stIt is possible for LE to appear in a variety of shapes  as well as a variety of sizes (1-10 mm in length and 1 mm in thickness) . It is pA study conducted by Roldan et al. and Cohen et al. found that LE may not be a cause of cardioembolic strokes, may be present in normal valves without any pathological process, and may not require treatment ,13. ThisTo date, no definitive treatment has been identified for LE. A patient's presentation will determine the type of treatment to be administered . AntiplaLambl's excrescence continues to be a very rare cause of cardioembolic stroke without an established treatment. Herein, we present a patient with an established stroke confirmed to have Lambl’s excrescence. A combination of medical and surgical management may be beneficial; however, more research is needed to determine the effectiveness of this approach.We present an exceptional case report of Lambl's excrescences (LEs) in a patient who had suffered an ischemic stroke. This singular case serves as an illustrative example, underscoring the requirement for further prospective investigations aimed at establishing a definitive association between LE and cardioembolic strokes. The importance of such research endeavors cannot be overstated, since LE has the potential to cause recurrent ischemic events leading to lasting disability and irreversible damage. The significance of elucidating this relationship lies in its potential to enhance our understanding of the pathophysiological mechanisms at play. This will also enable clinicians to develop targeted interventions and ultimately improve patient outcomes.


Phytosterols (PS) have been shown to regulate cholesterol metabolism and alleviate hyperlipidemia (HLP), but the mechanism is still unclear. In this study, we investigated the mechanism by which PS regulates cholesterol metabolism in high-fat diet (HFD) mice. The results showed that PS treatment reduced the accumulation of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the serum of HFD mice, while increasing the serum levels of high-density lipoprotein cholesterol (HDL-C). Compared with HFD mice, PS not only increased the antioxidant activity of the liver but also regulated the mRNA expression levels of enzymes and receptors related to cholesterol metabolism. The hypolipidemic effect of PS was abolished by antibiotic (Abx) intervention and reproduced by fecal transplantation (FMT) intervention. The results of 16S rRNA sequencing analysis showed that PS modulated the gut microbiota of mice. PS reduced the relative abundance of Lactobacillus and other bile salt hydrolase- (BSH-) producing gut microbiota in HFD mice, which are potentially related to cholesterol metabolism. These findings partially explain the mechanisms by which PS regulates cholesterol metabolism. This implies that regulation of the gut microbiota would be a potential target for the treatment of HLP. Since the 21st century, with the change of people's diet and lifestyle, obesity has gradually replaced malnutrition and infectious diseases as one of the most serious global epidemics causing medical problems . Currentβ-sitosterol, stigmasterol, campesterol, and brassicasterol [β-sitosterol, is widely studied and used, and it has been shown to be effective not only in lowering blood lipids but also in relieving colon inflammation and antianxiety [Phytosterols (PS), a class of plant-derived steroids that contain the same fused four-ring core structure  have a scasterol . The mosianxiety , 13. Stiianxiety . Campestianxiety . Since Pianxiety , 17. Henianxiety , 19.Although the benefits of PS affecting cholesterol metabolism have been reported constantly, the mechanism is still unclear. In this study, we used the HFD-induced HLP model to investigate the role of PS in regulating lipid metabolism. In addition, this study describes the role of PS in regulating the gut microbiota. We were attempting to explain the mechanism of PS in regulating cholesterol metabolism and the role of gut microbiota in this process.β-sitosterol content is more than 40%, campesterol is more than 20%, and stigmasterol is more than 14% . The purity of PS is more than 95%, 4% Table . High-faFive-week-old C57BL/6J male mice, weighing 18-20 g, were purchased from Beijing HFK Bioscience Co., Ltd. Animals were kept in the South China Agricultural University Laboratory Animal Center (SYXK 2019-0136) at a room temperature of 25 ± 2°C and relative humidity of 55 ± 5°C. All experiments were performed in accordance with animal welfare and the standards of the South China Agricultural University Experimental Animal Ethics Committee. All experiments were approved by the Ethics Committee.After 1 week of adaptive feeding, C57BL/6J male mice were randomly divided into three groups: normal diet group (ND), high-fat diet group (HFD), and high-fat diet with phytosterol group (HFD+PS). In the first 4 weeks, mice in the ND group were fed normal chow, while mice in the HFD and HFD+PS groups were fed high-fat chow at 25%, 50%, 75%, and 100%, respectively. From the 5th week, mice in the HFD+PS group were gavaged with 100 mg/kg of PS. Mice in the ND and HFD groups were gavaged with equal amounts of saline once a day for 8 weeks. Mice were executed at the end of the experiment. Samples were collected, and data were recorded according to experimental necessity.Twenty C57BL/6J male mice were adapted for 1 week and kept on high-fat chow for 12 weeks. At weeks 9 and 10, antibiotics  were added to the drinking water of mice in the HFD+Abx+PS group for clearing the gut microbiota. After that, both groups of mice were gavaged with 100 mg/kg/d PS for 2 weeks. At the end of the experiment, mice were executed, and tissues were collected.Ten C57BL/6J male mice were adaptation housed for 1 week. After 100 mg/kg/d PS gavage treatment for 2 weeks, 150 mg fresh feces of fecal microbiota transplantation (FMT) donor mice were collected into a sterile test tube every day, mixed with 0.9 mL PBS and suspended under anaerobic conditions, and vortexed for about 4 min. The mixture was centrifuged for 5 min at 4°C, 1000 × g, and the supernatant was aspirated as FMT material, gavaged to recipient mice immediately after preparation.Twenty C57BL/6J male mice were adapted for 1 week and kept on high-fat chow for 12 weeks. At first 2 weeks, antibiotics were added to the drinking water of mice in the HFD+Abx+FMT group to clear the gut microbiota. At the 3rd week, mice in the HFD+Abx+FMT group were conducted FMT, 0.2 mL/d for 1 week, while mice in the HFD+NS group were given with normal saline. At the end of the 12th week, animals were euthanized, and tissues were collected.Mice were fasted 12 hours in advance, and blood was collected from the tail vein using a blood collection needle. Blood glucose concentration was measured with a glucometer. Subsequently, mice were gavaged with 2.0 g/kg of glucose solution. The blood glucose concentrations of mice were measured after 30 min, 60 min, 120 min, and 180 min. The concentration curves were plotted, and the area under the curve (AUC) was calculated using GraphPad Prism 7.0 software.The liver and epididymal fat of mice were fixed in 10% formalin. Hematoxylin-eosin (H&E) staining was performed on conventional paraffin-embedded tissues (nuclei were blue and cytoplasm was red), and histological examination was performed using a light microscope at 200× visual field. Frozen liver sections were stained with oil red O solution and hematoxylin solution (lipid droplets are orange to bright red and nuclei are blue). Images were acquired for analysis at 200× visual field.https://www.cusabio.com/) according to the manufacturer's instructions.The blood was collected and centrifuged at 3000 r/min for 5 min, and then, the upper serum layer was separated and stored at -80°C. Serum levels of TC and TG were measured by ELISA kit, which was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd . Serum levels of LDL-C and HDL-C were determined using ELISA kits . The primers were synthesized by Tsingke Biotechnology Co., Ltd (China). The primer sequences are listed in Table Total tissue RNA was extracted using the RNA Isolater Total RNA Extraction Reagent kit (R401), and RNA was reverse transcribed to cDNA using the HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (R323). The reaction system was configured and performed according to the ChamQ Universal SYBR qPCR Master Mix kit (Q711), and the relative expression of target genes was analyzed by the 2DNA extraction from mouse fecal samples was performed using DNeasy PowerSoil Kit (Mo Bio/QIAGEN) according to the manufacturer's instructions. The absorbance values of DNA at 260/280 nm were measured using a fluorescence spectrophotometer to assess the concentration of sample DNA. The quality of DNA was detected by 1% agarose gel electrophoresis. Primer sequences were F: ACTCCTACGGGAGGCAGCA and R: GGACTACHVGGGTWTCTAAT. The V3-V4 region of the microbial 16S rRNA gene was amplified by PCR. Sequencing was performed by Shanghai Personal Biotechnology Co., Ltd. using Illumina MiSeq gene sequencing platform.T-test. Multiple data groups were compared using one-way ANOVA and Tukey's multiple comparisons to analyze the variability between groups. P < 0.05 were considered statistically significant. Correlation analysis was performed by the genes cloud tools (https://www.genescloud.cn).The experimental data were statistically analyzed using GraphPad Prism 7.0 software. Data comparison between two groups was analyzed by According to the experimental design of the animal protocol, we monitored the body weight of each group of mice weekly . CompareNext, to determine whether PS ameliorated glucolipid metabolism, we measured the serum concentrations of TG, TC, LDL-C, and HDL-C in mice, which are considered clinical biomarkers of severe HLP . The conNext, we examined the levels of oxidative stress-related mRNA in the liver. Compared with the ND mice, HFD significantly decreased the mRNA levels of heme oxygenase 1 (HO-1); compared with the HFD group mice, PS significantly increased the mRNA levels of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor, erythroid 2 like 2 (Nrf2), HO-1, NAD(P)H quinone dehydrogenase 1 (NQO1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC) Figures . These rThese results indicated that HFD significantly increased the concentrations of serum TG, TC, and LDL-C and decreased HDL-C, causing HLP and leading to impaired glucose tolerance in mice. PS treatment reversed these changes and ameliorated HLP and glucose tolerance.α-hydroxylase (CYP7A1), sterol 12α-hydroxylase (CYP8B1), oxysterol 7α-hydroxylase (CYP7B1), and sterol 27-hydroxylase (CYP27A1) and the bile acid (BA) synthesis rate-limiting enzyme related to cholesterol metabolism. It showed that compared with ND mice, the expressions of CYP7A1 and CYP8B1 were strongly increased in HFD mice, and the expressions of CYP27A1 and CYP7B1 also showed an increasing trend. PS intervention caused a significant decrease in the relative mRNA expression of CYP8B1 and a downward trend of CYP7A1 in HFD mice. CYP7B1 was significantly upregulated, and CYP27A1 was trended to increase in HFD+PS mice , Takeda G-protein-coupled receptor 5 (TGR5), and Farnesoid X receptor (FXR) and a decrease in the relative mRNA expression of small heterodimer protein (SHP) in mice. In HFD mice, PS treatment resulted in a significant decrease in the relative mRNA expression of TGR5 and FGFR4 and a significant increase in the mRNA levels of FXR and SHP in the liver Figures .As mentioned above, PS inhibited the classical pathway of hepatic cholesterol metabolism and promoted the expression of rate-limiting enzymes of alternative pathways. Meanwhile, PS inhibited TGR5 and FGFR4 and promoted the expression of FXR and SHP receptors.Previous studies have shown that the gut microbiota and its metabolites are closely related to cholesterol metabolism , 21. To Firstly, the results of principal component analysis (PCA) of gut microbiota showed that there were different clusters of gut microbial species abundance in the ND, HFD, and HFD+PS groups . Also, hHLP is a common and prevalent disease that occurs in obese or overweight people, and it is often complicated with diabetes, hypertension, and coronary heart disease , 23. TheWe hypothesized that the hypolipidemic effect of PS is related to the pathway of cholesterol metabolism in the liver. BA metabolism is an important route of cholesterol excretion. About one-third of the cholesterol catabolism in the body is achieved by BA synthesis . The entAccording to the previous reports, signaling by FXR and FGFR4 receptors in the liver can control BA synthesis pathways in the liver, while BA transport in the enterohepatic cycle alters the composition, size, and distribution of the BA pool, which in turn can have a significant impact on BA signaling and its downstream metabolic targets . FXR is Lactobacillus, Lactococcus, Streptococcus, Bacillus, and Bifidobacterium, and the highest enzymatic activity was reported for BSH-T3 present in Lactobacillus [Lactobacillus reduces BSH activity and leads to changes in the composition of BA, ultimately alleviating obesity [Lactobacillus, Lactococcus, Streptococcus, and Lactobacillus vaginalis was strongly associated with hyperlipidemia; also, these genera and the expression of CYP7A1 and CYP8B1 were positively correlated, while Bacillus was negatively correlated with CYP7B1. Our analysis of the gut microbiota showed that PS reduced the relative abundance of Lactobacillus and other BSH-producing gut microbiota in HFD mice, which is probably critical for regulating BA metabolism [Lactobacillus vaginalis. Furthermore, Allobaculum was thought to be positively correlated with HLP, and this was reinforced by the fact that PS intervention resulted in a significant reduction of Allobaculum in HFD mice [As mentioned above, BA synthesis is an important pathway of cholesterol metabolism. Signaling in the hepatic-intestinal axis directly regulates the relationship between gut microbiota and BA metabolism , 55. In bacillus . In cont obesity . Previou obesity . In thistabolism , 62. IntHFD mice , 64.In previous studies, the mechanisms of cholesterol absorption inhibition by PS are mainly focused on competition for binding sites, reduction of cholesterol absorption, and interference with BA metabolism mechanisms , 66. SomIn summary, we found that PS significantly increased the antioxidant function of the liver and alleviated HLP in mice. The mechanism was related to the regulation of cholesterol metabolism and the structure of the gut microbiota. The relative abundance of the gut microbiota is regulated when treated with PS. SHP and FXR were activated, and FGFR4 and TGR5 were inhibited, while the classical pathway of BA synthesis (CYP7A1 and CYP8B1) was inhibited, and the alternative pathway of BA synthesis (CYP27A1 and CYP7B1) was activated treated with PS. Our results partially explain the mechanism of PS treatment of HLP, which may be related to the regulating cholesterol metabolic pathways and the structure of the gut microbiota.


How back pain is related to intervertebral disc degeneration, spinal loading or sports-related overuse remains an unanswered question of biomechanics. Coupled MBS and FEM simulations can provide a holistic view of the spine by considering both the overall kinematics and kinetics of the spine and the inner stress distribution of flexible components. We reviewed studies that included MBS and FEM co-simulations of the spine. Thereby, we classified the studies into unidirectional and bidirectional co-simulation, according to their data exchange methods. Several studies have demonstrated that using unidirectional co-simulation models provides useful insights into spinal biomechanics, although synchronizing the two distinct models remains a key challenge, often requiring extensive manual intervention. The use of a bidirectional co-simulation features an iterative, automated process with a constant data exchange between integrated subsystems. It reduces manual corrections of vertebra positions or reaction forces and enables detailed modeling of dynamic load cases. Bidirectional co-simulations are thus a promising new research approach for improved spine modeling, as a main challenge in spinal biomechanics is the nonlinear deformation of the intervertebral discs. Future studies will likely include the automated implementation of patient-specific bidirectional co-simulation models using hyper- or poroelastic intervertebral disc FEM models and muscle forces examined by an optimization algorithm in MBS. Applications range from clinical diagnosis to biomechanical analysis of overload situations in sports and injury prediction. When humans evolved to adopt an upright position, the spine became a central structure of the human biomechanical system. As such, it can be a source of pain that can significantly impact a person’s quality of life. Intervertebral disc (IVD) degeneration and herniation are possible causes of back pain. Research has shown that both aging  and overMultibody simulation (MBS) is widely used to gain insights into the healthy and pathological biomechanics of the spine from a macroscopic perspective. MBS models study joint reaction forces, muscle forces and muscle activation patterns in combination with respective movements or positions. There are several such models that include intervertebral joints with three rotational ,17,18,19Finite element method (FEM) spine models aim to simulate deforming bodies in a detailed manner to reveal their inner stresses. Models may include the whole spine, a segment or only an IVD. Most models contain manually created geometries for IVDs and vertebrae , but recIn summary, MBS models are suitable for investigations of the overall kinematics and kinetics of the trunk, or even the full body. However, detailed information on flexible body deformation and internal stress cannot be provided. Complex, heterogeneous structures, such as the IVD, or even entire functional spinal units (FSU) combining all stabilizing structures , are reduced to a resultant mechanical response to external deformation, often in reference to an approximated center of rotation. In contrast, FEM models are beneficial for simulating detailed structures and analyzing inner stresses and deformations. However, detailed FEM meshes may cause large computational costs, and thus, models often include simplifications. Additionally, defining realistic boundary conditions (BC) for FEM models remains a main challenge due to the lack of possibilities for in vivo measurements. Until now, no satisfactory, broad investigations are available containing this data .Coupling MBS and FEM takes advantage of the strengths of each method and offers great potential to analyze the deformations and stresses of IVDs while considering the overall kinematics and kinetics of the trunk. How coupling is implemented can be subdivided into two approaches: One is to calculate acting forces or present deformations completely before starting the other simulation . The second approach considers acting forces or displacements anew in every increment of an ongoing simulation, generating a constant, bidirectional data flow. Unidirectional coupling commonly includes an inverse dynamic MBS simulation with resulting forces and moments, which are subsequently used as loading and BCs in an FEM model ,41,42,43Hence, by using co-simulation, we can examine the pathological spine in a holistic way to analyze the interactions of various factors, such as motion, posture, back pain and the biomechanics of the IVD. In this work, we review advances in coupled MBS and FEM simulations of the spine. Specifically, we excluded reduced order models and instead focused on the type of data exchange, whether unidirectional or bidirectional, as this is essential for the simulation of large deformations and constant interactions of different components in the spine.Coupling MBS and FEM models has become a common practice in spine modeling during the last decade. However, no terminology has been established yet to differentiate between what are here called unidirectional and bidirectional co-simulation. To achieve comprehensive coverage of studies using co-simulation, the following keywords were searched in several combinations in different databases: coupled, spine, multibody, MBS, FEM, hybrid, finite element, co-simulation, combined. Resulting articles were manually reviewed and included only if they: (i) contained a type of coupling between MBS and FEM and (ii) were published after 2009 or included models that were still used in studies published after 2009.We first reviewed MBS and FEM models of the spine using unidirectional coupling, which was characterized by the execution of one simulation and the integration of the results into another simulation. Considering the order of execution, three distinct methods of coupling were identified: MBS execution and transfer of data to an FEM simulation; FEM and transfer of data to MBS; and a threefold data transfer from FEM to MBS and to FEM again. A unidirectional data transfer from MBS to FEM was the most common approach in unidirectional coupling. Muscle forces, reaction forces or displacements were calculated by an MBS simulation and integrated as boundary conditions (BC) in a subsequent FEM simulation.Esat et al. ,38 estabDu et al.  investigHenao et al.  simulateHonegger et al.  investig(I)The first model included an initial, detailed FEM representation of the spine.(II)The second model was based on the detailed model but consisted of rigid bodies and interconnecting beam elements. It is later referred to as the musculoskeletal (MS) model.A research group bulit around Shirazi-Adl and Arjmand has been developing numerical models of the spine since 1985  and has Version II was not solved with a classic MBS solver, but included clear characteristics of an MBS model, such as the involvement of mainly rigid bodies and their interconnections by joint-like components. While version II could not represent detailed deformations, it was able to perform large numbers of nonlinear analyses . The resIn 2002, Shirazi-Adl et al.  incorporAzari et al.  modifiedm and reaction moment MCoR, for each muscle. Reaction forces were considered as the sum of gravitational forces and muscle forces. The resulting forces and moments in a static equilibrium condition calculated in the MS model were applied to the vertebral growth plate FEM bodies as distributed pressures and shear stresses. Note, IVDs were solely considered as joints, and the biomechanical focus lay on the growth plates. FEM simulations were carried out with Abaqus . The resulting stresses in the growth plates were comparable with results from the literature and may be helpful in predicting growth patterns in scoliotic spines.To analyze scoliotic spine loads and their growth patterns, Kamal et al.  establisFurther studies included a hybrid model applying muscle forces calculated in the beam-joint-based MS model (II) to the updated, passive T12-S1 FEM model (I) . The MS In order to develop a self-defined gold standard of spine modeling, Rajaee et al.  further To sum up, multiple simulation approaches have been executed using MBS simulation results in FEM models. Transferred data included reaction forces, muscle forces or displacements of vertebrae on different spine levels. The vast majority of MBS studies rely on experimental data to define joint properties of the IVDs. However, one study investigated the potential of FEM simulations to define MBS model properties.Karajan et al.  investigThe resulting MBS model with adapted stiffness parameters could potentially be further used to provide information for a detailed FEM simulation, calculating stresses and deformations. This step has been realized by some research groups. Studies are reviewed in the following.To investigate the effects of muscle damage as seen in lumbar interbody fusion surgery, Kumaran et al.  used mulIn 2021, Meszaros et al., presented a study in which they adapted an established neuro-musculoskeletal spine model  to matchR line of action until it reached to MBS-predicted position. The novel reaction force RF obtained was iteratively compared with the initial reaction force FR while adjusting the translation, until the difference was smaller than a predefined tolerance. In a follow-up work, Liu and El-Rich used the same model, reduced to one functional spinal unit (L4–L5), to investigate the influences of the NP position on the IDP, spinal loads and load sharing during 60° forward flexion. Based on in vivo data, three posterior shifts of the NP were realized by the models: 0, 1.5 and 2.7 mm. Muscles and ligaments forces, and joint forces and moments at L3–L4 were calculated by the MBS model and prescribed to the FEM model. IDP and spinal loads calculated by the FEM model show that the IDP and compressive forces within an FSU were distinctly influenced by the posterior shifts of the NP, and the CoRs calculated by their MBS and FEM model differ. Liu and El-Rich believe the kinetic results predicted by the MBS model to have been affected by single IVD rotating joints and suggest implementing an iterative process combining MBS and FEM models to account for compressive and shear stiffness. [Load sharing in the lumbosacral spine was explored by Liu et al.  in 2018 iffness. Refer to Independent of the data transfer being solely from MBS to FEM, from FEM to MBS or both, unidirectional co-simulation was often limited by linear deformations and manual adaption processes to synchronize both models ,40,42. TBidirectional co-simulation models benefit from an iterative data exchange that requires less manual intervention and more accurately accounts for large deformations of, e.g., IVDs. Approaches using this type of coupling are listed below, again focusing on how data are exchanged between MBS and FEM models.Monteiro et al.  realizedAnother coupling method was implemented by Dicko et al.  in combiIn 2021, Remus et al.  publisheRefer to Most co-simulation models of the spine use a unidirectional coupling approach, transferring data singularly from one simulation to the other by applying both simulation methods consecutively. MBS spine models can profit by defining joint stiffness parameters based on FEM simulations of the IVD. FEM models of the spine or spinal components can be improved when muscle or ligament forces and moments are implemented as BCs. However, when providing a time-dependent input, the time-dependency is influenced by the deformation properties of a material or component. Equal mechanics cannot be expected when comparing FEM and MBS IVD representations due to their different modeling approaches. The input accomplished by the results of the MBS initially carried out is therefore only partly suitable for being incorporated in a subsequent FEM simulation. This limitation is demonstrated in the effort that has been put into synchronizing the respective models of the unidirectional co-simulations. The manual adaption steps that become necessary may provoke inaccuracies and take much time. Kumaran et al. , for exaTo overcome these limitations, a few recent studies have implemented bidirectional co-simulations of the spine. By updating the deformation values of the IVDs and the resulting positions of the vertebrae in every increment, updated reaction moments and forces of muscles, tendons and ligaments can be considered. Of the research groups using bidirectional co-simulation models, all authors found that their models were able to deliver accurate results ,47,73. HThe particle-based approach divides the whole model into two types of particles, rigid and flexible ones ,73,74. TDespite these limitations, bidirectional co-simulation models of the spine can provide a holistic understanding of the spine because they consider both the overall kinematics with the muscular and gravitational forces and moments, and the detailed mechanics of the IVDs with their deformations and stress distributions.FEM is broadly used in detailed analyses of internal stresses in deforming bodies but lacks computational efficiency. MBS is more efficient, but can only achieve a certain degree of detail when it comes to deformations of model components. A combination of both methods, not only in a unidirectional way, but in a bidirectional manner of data exchange, can provide both accuracy and efficiency.Future studies will likely include widespread use of bidirectional co-simulation models to understand and predict the behavior of the spine. Including automated segmentation algorithms such as the one implemented by Sekuboyina et al.  could ac


Disordered urban environments negatively impact mental health symptoms and disorders. While many aspects of the built environment have been studied, one influence may come from inequitable, discriminatory housing practices such as redlining, blockbusting, and gentrification. The patterns of disinvestment and reinvestment that follow may be an underlying mechanism predicting poor mental health. In this study, we examine pathways between such practices and internalizing symptoms  among a sample of African American youth in Baltimore, Maryland, considering moderation and mediation pathways including neighborhood social cohesion and sex. In our direct models, the inequitable housing practices were not significant predictors of social cohesion. In our sex moderation model, however, we find negative influences on social cohesion: for girls from gentrification, and for boys from blockbusting. Our moderated mediation model shows that girls in gentrifying neighborhoods who experience lower social cohesion have higher levels of internalizing symptoms. Likewise for boys, living in a formerly blockbusted neighborhood generates poorer social cohesion, which in turn drives higher rates of internalizing symptoms. A key implication of this work is that, in addition to standard measures of the contemporary built environment, considering other invisible patterns related to discriminatory and inequitable housing practices is important in understanding the types of neighborhoods where anxiety and depression are more prevalent. And while some recent work has discussed the importance of considering phenomena like redlining in considering long-term trajectories of neighborhoods, other patterns such as blockbusting and gentrification may be equally important. Internalizing symptoms (including anxiety and depression) are relatively common across adolescence , but minWhile redlining was outlawed in 1968, the decades-long practice of excluding minorities from access to mortgages created huge gaps in accrued wealth. The ensuing practice of blockbusting and white flight—generated by the mortgage and banking industry in the 1970s and 1980s to spark panic selling and “flip” previously all-white neighborhoods —led to aDisinvested urban environments trend with higher rates of anxiety and depression among youth , though In the current study, we examined: (a) whether inequitable housing practices—specifically redlining, blockbusting, and gentrification—were associated with internalizing symptoms, (b) whether these practices predict levels of neighborhood cohesion for boys and girls, and (c) whether neighborhood social cohesion mediated the relation between these neighborhood practices and internalizing symptoms. We examine all three practices in this article because the associations between the processes and outcomes are likely distinct, and merit consideration.Such practices are essential to consider and correct because the decades-long processes of structural racism in housing make us more vulnerable to the impacts of climate and urban change . America1.1.1.1.1.Redlining and blockbusting are two inequitable housing practices that have upheld racial and economic residential segregation and are responsible for vast differences in the quality of the built environment. Prior to 1968, no federal law ensured fair housing for all races . StartinFollowing redlining’s prohibition, blockbusting was used to maintain segregation. Real estate agents and the mortgage industry colluded to create panic selling in previously all-white neighborhoods, convincing white residents to sell low and re-selling these homes at a premium to African American and other minority families . BlockbuIn addition to the direct financial implications of housing discrimination , it also1.1.2.Gentrification commonly entails an in-migration of middle-income residents, while spurring displacement of lower-income residents . It ofteA number of studies have linked gentrification with myriad health outcomes, although findings are mixed . GentrifHowever, there is also evidence that gentrification may contribute to better health outcomes. For example, increased empowerment for community improvement and cross-cultural exchange may bring new opportunities . Moreove1.2.Inequitable housing practices uphold racial and economic residential segregation and influence unequal access to resources and treatment  that creWhile segregation may have a protective effect on mental distress via living among one’s own group , it typiPeople in segregated or resource-scarce neighborhoods may also internalize these environments as a personal deficit instead of seeing the structural racism that caused it, which may contribute to feelings of depression and anxiety. Conversely, racist societal assumptions contribute to the racial empathy gap and implicit bias that is negatively experienced among minority populations. Ethnic density can lessen depressive symptoms to a point but is shown to contribute to higher levels in highly segregated neighborhoods . AdditioWhere new investments do take place (often in areas considered to be gentrifying), the resulting housing inequalities may confer risk for internalizing symptoms among existing youth. For example, research suggests that more affluent older adults reported higher levels of anxiety and depressive symptoms relative to adults living in more economically depressed areas which may be due to concerns over increases in the cost of living, and anxiety regarding housing displacement and closure of businesses .1.3.Neighborhood social cohesion is defined by one’s sense of community, neighborly trust, and the positive social interactions that occur therein . While hIndeed, research suggests that social connectedness may buffer against the negative impacts of gentrification, particularly among vulnerable populations , althoug1.4.While it is possible that historical neighborhood practices and perceived neighborhood cohesion may impact internalizing symptoms, there is reason to believe that sex differences may impact these relations. For example, living in a low-income neighborhood has been shown to predict social anxiety for girls, but not boys . AdditioIn addition, girls may be impacted more by their environments than boys . For exa1.5.To address the existing gaps in the literature, the current study examined pathways between inequitable housing practices and internalizing symptoms and the differential impact of these effects on boys and girls. We hypothesized that (a) there would be a direct and positive effect of inequitable housing practices on internalizing symptoms, (b) housing practices would also predict neighborhood cohesion and that these relations would be further moderated by sex, and, finally, (c) neighborhood cohesion would mediate the relation between housing practices and internalizing symptoms in both boys and girls.2.Participants were predominantly African American youth from Baltimore, Maryland, originally recruited for the Youth Opportunity (YO) program. The YO program’s goals are to increase access to educational, occupational, and training opportunities for adolescents and young adults . The YO Mage = 18.76, SD = 1.71). Given the very small percentage of the sample that was not African American, only African Americans were included in the analyses . Approximately 60% of the sample participated in one of the YO programs and about 10% of the sample reported being employed (see Data were collected at three time points: baseline (when the study began in 2008), six months post-baseline, and one to two years post-baseline. Baseline data for the current study included 782 youth , reverse coded, and summed with higher scores reflecting higher levels of social cohesion. In the current sample, the measure demonstrated excellent internal reliability .We assessed neighborhood social cohesion using a three-item scale developed by 2.1.2.For each participant, we joined variables denoting whether they lived in an area that had been redlined, blockbusted, or gentrified  and summed to create a composite score (α = 0.90). The measure demonstrated excellent internal reliability in the current sample .Internalizing symptoms were assessed by creating a composite of measures of anxiety and depressive symptoms. Anxiety symptoms were evaluated using the BAI . The BAIrarely or none of the time to 4 = most or all of the time) and summed to create a composite score (α = 0.86). In the current sample, the measure demonstrated internal reliability . The anxiety and depression composites were z-scored  and summed to create an internalizing symptom composite .Depression symptoms were assessed using the CES-D . The CES2.2.Patterns of missing data and univariate normality were examined for all variables. Means and standard deviations between key study variables were also evaluated. Inequitable housing practices were examined in separate models to reflect the distinct time periods and investment patterns of each practice. Each of the hypotheses regarding the effects of inequitable housing practices was tested in a series of main effect and mediation models. First, the direct effects of sex, age, and each of the three inequitable housing practice predictors  on internalizing symptoms were evaluated. Second, the main effects of housing practices on neighborhood cohesion were examined. Next, we examined whether these relations were moderated by sex. Finally, we evaluated a moderated mediation model in which we examined whether the indirect pathway from inequitable housing practices to internalizing symptoms via neighborhood cohesion differed for boys and girls see .All analyses were run in SPSS Version 24 using the PROCESS macro. Non-parametric bootstrapping procedures  were utilized to evaluate the significance of the indirect effects as well as examine an index of moderated mediation. Unlike hypothesis testing based on parametric statistics, bootstrapping procedures do not assume that the indirect effect (the product of the effect of the independent variable to the mediator and the effect of the mediator on the outcome) is normally distributed . Indirec3.r = −0.39) and gentrification (r = −0.31) were moderate and negative, while the relation between redlining and gentrification was moderate and positive (r = 0.35). Results for the primary analyses are presented below.Very low rates of missing data were found for each variable (0–3.7%). All dependent variables were found to be within acceptable ranges for skew and kurtosis (≤3.0). The correlations between blockbusting and both redlining  in three separate models. Across all models, only sex and age were significant predictors of internalizing symptoms, which indicated that girls and older youth experienced greater levels of symptomatology.3.2.In our second set of models, we examined inequitable housing practices as predictors of neighborhood cohesion in three separate models. In each of the models, only younger age was consistently linked to greater perceived neighborhood cohesion. Sex was also associated with neighborhood cohesion, indicating that boys reported higher levels of perceived neighborhood cohesion. None of the inequitable housing practices was a significant predictor of cohesion.3.3.β = −0.233, p = 0.050), such that there was a negative effect of gentrification on neighborhood cohesion for girls only ; however, this effect was in the opposite direction. Results suggest a significant, negative effect of blockbusting on neighborhood cohesion for boys only, indicating that boys who lived in areas with higher rates of blockbusting reported less neighborhood cohesion . The first model found a marginally significant interaction between gentrification and sex . Moreover, the difference between the indirect effects for boys and girls was statistically significant . These findings indicate girls exposed to higher rates of gentrification experienced lower neighborhood cohesion which, in turn, predicted elevated levels of internalizing symptoms . Results support a moderated mediation model, indicating a significant indirect effect of gentrification on internalizing symptoms for girls only . The index of moderated mediation was also significant  suggesting these differences were statistically significantly different between boys and girls.Our next set of models evaluated a mediation model in which exposure to blockbusting predicted neighborhood cohesion which, in turn, predicted internalizing symptoms (controlling for participant age and sex). Findings suggest that the direct effect of blockbusting on internalizing symptoms was not significant, nor was the indirect effect via neighborhood cohesion. We then added sex as a moderator of the pathway from blockbusting to neighborhood cohesion within the larger mediation model (continuing to control for participant age). Results indicate that a significant interaction effect between sex and blockbusting predicting neighborhood cohesion in the same pattern as reported above. Findings suggest a significant moderated mediation effect, indicating that living in a historically blockbusted area was associated with lower neighborhood cohesion for boys only and that this, in turn, predicted higher rates of internalizing symptoms , we found no direct or indirect effect of redlining on internalizing symptoms. We then considered a moderated mediation model (controlling for participant age). We found that sex did not moderate the pathway from redlining to neighborhood cohesion and that there was not a significant moderated mediation effect. All moderated mediation models were also run controlling for each of the other housing practices. An identical pattern of results emerged.4.Our first major finding is that girls living in gentrifying neighborhoods reported lower perceived neighborhood cohesion, which in turn predicted elevated levels of internalizing symptoms. The fact that girls in gentrifying neighborhoods experienced greater levels of internalizing symptoms suggests that neighborhoods may fail to incorporate some existing residents into the new and changing social life of the community, which contributes to the development of internalizing symptoms, particularly among girls.A second major finding was that boys living in previously blockbusted neighborhoods reported less neighborhood cohesion which in turn predicted higher rates of internalizing symptoms. Blockbusted neighborhoods are effectively places of severe white flight and disinvestment . That boOur analyses did not find any impacts of redlining on social cohesion or internalizing symptoms. Although the impacts of redlining on inter-generational wealth and other issues remain unresolved, the lack of an association suggests that people physically living in these spaces do not experience significantly worse outcomes than people in other neighborhoods. And while redlining and gentrification were coincident in some cases , blockbuGiven the significant effects of blockbusting on health outcomes, these findings are important; they illustrate the need for land use policies that address legacy effects of types of housing discrimination beyond redlining. Such understanding is essential for future urban planning approaches that aim to build more equitable cities.4.1.Despite these strengths, our findings should be considered in light of a few limitations. First, the study utilized a cross-sectional design, which prevented us from disentangling the temporal relations between neighborhood cohesion and internalizing symptoms. It is possible that youth who experience higher levels of internalizing symptoms may, in turn, experience their neighborhoods as less interconnected. Second, we utilized self-report assessments to capture individuals’ perceptions regarding both neighborhood characteristics and internalizing symptoms, which may have introduced bias related to shared method variance. Subsequent research into these domains may consider using other methods to evaluate neighborhood cohesion, including social network analyses, to more objectively capture these relations. Third, few studies have used an operational GIS-based definition of blockbusting as in . While t4.2.As the fields of public health and urban planning continue their path toward reconnection , we alsoSpecifically, our use of GIS to connect individual participants’ neighborhoods to inequitable housing practices is particularly novel. This approach allowed us to capture objective measures of historical neighborhood characteristics and examine the influence of participants’ perceptions of neighborhood cohesion and mental well-being. Additional strengths of the study include the careful examination of the role of participant sex in study constructs. While other studies suggest that geographic characteristics may impact boys’ and girls’ mental health differentially , this isThese results have potential policy applications, as they demonstrate the impacts of decades of housing practices on mental health outcomes. These findings highlight the need for considering mental health in determining housing policy  and sugg